Browsing by Subject "Amino acids"
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Item A study of the amino acids associated with ovalbumin(Texas Tech University, 1970-12) Hicks, John EdgarNot availableItem An amino acid mixture enhances insulin-stimulated glucose uptake in isolated epitrochlearis muscle(2010-08) Kleinert, Maximilian; Ivy, John, 1945-; Farrar, Roger P.Amino acids are important modulators of skeletal muscle metabolism, but their impact on glucose uptake by skeletal muscle remains unclear. To address the effect of an amino acid (AA) mixture consisting predominately of isoleucine on glucose uptake we first conducted a dose-response experiment, investigating how different concentrations of the AA mixture affect glucose uptake by isolated rat epitrochlearis muscle. In a subsequent experiment we examined how the AA mixture affects insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle. It was found that the AA mixture with as little as 0.5 mM Ile increases [H3]2-deoxy-D-glucose (2-DG) uptake by 76% compared to basal glucose uptake. The AA mixtures with 1, 2 or 4 mM Ile provided no significant additional effect. Next we combined the AA mixture consisting of 2 mM Ile, 0.012 mM Cys, 0.006 mM Val and 0.014 mM Leu with physiological levels (75 μU/ml, sINS) and maximally-stimulating levels (2 mU/ml, mINS) of insulin. The AA mixture only, sINS and mINS significantly increased 2-DG uptake compared to basal by 63, 79 and 298%, respectively. When the AA mixture was combined with sINS and mINS 2-DG uptake was further increased significantly by 26 and 14%, respectively. Western blotting analysis revealed that compared to basal the AA mixture increased AS160 phosphorylation, while phosphorylation of Akt and mTOR did not change. Combining the AA mixture with sINS resulted in no additional phosphorylation compared to sINS alone. Interestingly, addition of the AA mixture to mINS resulted in increased phosphorylation of mTOR, Akt and AS160 compared to mINS alone. Our results suggest that certain AAs (1) increase glucose uptake in the absence of insulin and (2) augment insulin-stimulated glucose uptake in an additive manner. These effects on glucose uptake appear to be mediated via a molecular pathway that is partially independent from the canonical insulin signaling cascade.Item Amino acid nutrition and ideal protein for reproductive sows(Texas Tech University, 2004-12) Ji, FeiThe purpose of raising sows is to produce healthy weaning pigs. Sows should be maintained in healthy, strong, and good condition. High-prolific lean type sows have large litter size and high milk production, but low appetite during lactation, and therefore, high culling rate. Providing accurate amounts and profiles of amino acids to gestating and lactating sows will allow more efficient sow production and reduce feed cost and N excretion. Currently, there are some problems in sow nutrition: 1) current available amino acid requirements for gestating sows were adapted from growing pig data; 2) dietary amino acid ratios for the efficient protein utilization are not known for sows; and 3) current amino acid requirements may not provide accurate estimations for gestating and lactating sows. The current feeding system for gestating sows (one drop, single diet) is not sufficiently flexible to adjust the nutrient allowance according to the nutritional status of sows. Therefore, the amino acid requirements and ideal dietary amino acid ratios for gestating and lactating sows need to be better characterized. This dissertation covers the areas of: 1) changes of body weight, backfat thickness, and chemical contents in various maternal and fetal tissues in pregnant gilts and estimation of protein needs in different gestation stages of gilts; 2) growth of mammary glands of gilts during gestation; 3) estimation of ideal protein for pregnant gilts; 4) validation of ideal protein for pregnant gilts; and 5) validation of ideal protein for lactating sows. In the first study, 35 gilts were randomly allotted to seven slaughter groups: d 0, 45, 60, 75, 90, 102, and 112 of gestation. Gilts were bred and fed 2 kg/d gestation diet (as fed basis) during gestation. The gestation diet contained 3.1 Mcal/kg ME and 0.56% lysine (as fed basis). Maternal tissues and organs and fetuses were separated and weighed. Compositions of maternal and fetal tissues were analyzed. The protein accretion rates in various tissues increased (P < 0.05) after d 70 of gestation. Considering the needs of maintenance and maternal and fetal gains, we suggest that the diet of pregnant gilts should provide 6.8 and 15.3 g/d true ileal digestible lysine, respectively, before and after d 70 of gestation. Feeding gestating gilts based on a two-phase feeding program reflects the real protein needs for tissue protein accretion that may improve protein utilization. In the second study, individual mammary glands were separated and weighed. The first two pairs of glands were pooled as "anterior." The 3rd, 4th, and 5th pairs of glands were pooled as "middle." The 6th, 7th, and 8th pairs of glands were pooled as "posterior." The CP content in middle accreted faster than that in anterior and posterior. At d 112 of gestation, the CP content in individual mammary glands in middle was heavier (P < 0.05) than that in anterior and posterior. This result was different from the generally recognized concept that anterior mammary glands are larger and produce more milk than middle and posterior mammary glands. In the third study, based on changes of CP in various maternal and fetal tissues during gestation, ideal amino acid ratios for protein accretion and protein accretion plus maintenance for pregnant gilts were estimated. Amino acid needs of pregnant gilts for maintenance and amino acid accretion rates in maternal and fetal tissues changed during gestation; therefore, lysine-based amino acid ratios also changed. The ideal amino acid ratios were different in the early and late gestation. In the fourth study, thirty-four pregnant gilts at d 30 of gestation were used in two experiments. In Exp. 1, 14 pregnant gilts were randomly allotted to a control group (Cl, seven gilts) and an ideal protein group (IPl, seven gilts). In Exp. 2, 20 pregnant gilts were randomly allotted to a control group (C2, ten gilts) and an ideal protein group (IP2, ten gilts). Pregnant gilts had two meals of gestation diet (2.0 kg/d, as fed). The IPl was formulated to provide an ideal dietary amino acid ratio for maternal and fetal tissue gains, whereas the IP2 diet for both tissue gains and maternal maintenance. In Exp. 1, performance of gestation and subsequent lactation did not differ between the IPl and Cl. In Exp. 2, body weight gain of the IP2 was higher (P < 0.01) than that of the C2 fi'om d 30 to 109 of gestation. Serum urea level of the IP2 was lower (P < 0.05) than that of the C2 at d 90 and 109 of gestation. Litter mortality of the IP2 was lower (P < 0.05) than that of the C2 during lactation. Performance of the IPl was not improved, suggesting that the ideal amino acid ratios for protein accretion were not balanced for pregnant gilts. The IP2 had higher body weight gain (P < 0.01) from d 30 to 109 of gestation and lower serum urea level at d 109 of gestation (P < 0.05), suggesting that ideal amino acid ratios in the IP2 decreased the oxidation of amino acids and increased maternal protein accretion. In the fifth study, 51 sows were grouped according to their body weight at farrowing and parities. Within each group, sows were randomly allotted to four dietary treatments: 1) low-protein control (17.5% CP; LPC); 2) low protein with ideal protein (LPI); 3) high-protein control (19.5% CP; HPC); and 4) high protein with ideal protein (HPI). Ideal protein for lactating sows was obtained from the TTU-Sow Model (Kim et al., 2002). Feed intake and body weight loss of sows did not differ among four dietary treatments. Sows fed ideal protein had increased (P < 0.05) litter weight gain overall three parity at the low-protein diets. Sows fed ideal protein had decreased (P < 0.01) serum urea concentration at the high protein level. Sows fed ideal protein had increased (P < 0.05) litter weight gain of the first parity sows at the low-protein level. Sows fed ideal protein improved litter weight gain in the low-protein diet during lactation, indicating that consideration of ideal dietary amino acid ratio in lactation diet may enhance milk production. The benefits may be maximal for the first-parity sows because they have lower voluntary feed intakes and limited body tissue reserves. Current available estimation of amino acid needs of gestating and lactating sows for maintenance was based on the data of non-pregnant gilts. Whether pregnancy and lactation affect amino acid needs of gestating and lactating sows for maintenance is unknown. Further research is needed in these areas of nutritional physiology. In addition, we assumed that the metabolic rates of various amino acids in the small intestinal mucosa were similar between nonpregnant and pregnant pigs or between d 1 and d 21 of lactation, but some studies form other species suggested that the rates of amino acid synthesis and/or catabolism in enterocytes may vary with physiological status.Item Amino acid supplementation of grain sorghum for young pigs(Texas Tech University, 1971-08) McManigal, James GunterNot availableItem Aminopeptidases of Bacillus subtilis(Texas Tech University, 1975-05) Desmond, Edward PaulThe production of aminopeptidases, intracellularly or extracellularly, by many bacterial species has been noted (4, 5, 28). In the genus Bacillus, Aubert and Millet (2) reported an aminopeptidase in cells of _B. megaterium which increased five-fold in activity during sporulation. Hall, Kunkel and Prescott reported an extracellular aminopeptidase in culture filtrated of B. licheniformis which has a pH optimum of 8.5 - 9, and was activated by Co ions (15). Two aminopeptidases haye also been reported in B^. stearothermophi 1 us (25, 29). In B. subtilis, Matsumura, et al_., (23) and ainamiura, et al., (24) found two aminopeptidases in cells of this species which could be separated from each other on columns of diethylaminoethyl (DEAE) cellulose. Minamiura, ejt aj, reported that one of these two enzymes hydrolyzed D-leucyl-glycyl-glycine. Wagner, Chung, and Ray (32) described an extracellular aminopeptidase in cultures of B. subtilis which they contend is secreted, under appropriate conditions, by intact cells into the medium. The pH optimum of this enzyme was 8.0, and Co was found to activate it.Item Cation/amino acid symports in the photosynthetic bacterium Chromatium vinosum(Texas Tech University, 1984-05) Cobb, K. AndreaNot availableItem Characterization of an ORF in a novel orientation in Escherichia coli(Texas Tech University, 2003-12) Lackey, Melinda K.Evidence has been presented in the literature that demonstrates the expendable nature of the C-terminal region for the proper functioning of TolC in the bacterial cell. Recent sequencing of various microbial genomes demonstrated the presence of 3' overlapping, converging open reading frames involving tolC and its homologues found in other species and genera of bacteria. In this study, we examined the Escherichia coli tolC gene and the 3' overlapping, converging ORF found at the same genetic locus, denoted cloT for tolC in reverse. The expression of c/or was examined using reverse transcriptase PCR to identify the presence of mRNA molecules within the bacterial cell. Amplification of a 120 base pair fragment from total RNA isolated from wild-type E. coli suggested that cloT was transcribed, at least at low levels, in the organism. To corroborate these data, the upstream region of the structural gene was analyzed for the ability to drive transcription of a reporter gene, beta-galactosidase. Activity analysis of this reporter fusion demonstrated the ability of the upstream region of the cloT gene to drive transcription in E. coli. Similarly, we have demonstrated an interaction between the upstream region of cloT and soluble proteins isolated from the cytoplasm of wild-type E. coli. Further experiments were attempted to raise antibodies against a portion of the putative CloT protein using both GST fusion technology and a synthetic peptide. Neither has been fully characterized at time of publication. In addition, attempts to isolate mutations in the unique region of the cloTORF in the chromosome of E. coli were also unsuccessful. Examination of the information known about TolC and its homologues in different genera of bacteria revealed interesting information concerning the C-termini of these proteins. Alignment of the amino acid sequences demonstrated lower identity shared in the C-terminal region of these proteins. In addition, examination of the nucleotide sequences of these homologues indicated the presence of a similar open reading frame sharing a convergent, overlapping relationship with its respective tolC homologue. When combined with published data concerning the expendable nature of the last fifty amino acids of TolC, these data suggest that the presence of a conserved open reading frame in this region may play an important role in the regulation or function of the tolC gene or gene product(s).Item Design, synthesis & thermodynamic evaluation of conformationally-constrained pseudopeptides and synthetic approaches to sieboldine A(2008-08) Teresk, Martin Gerald, 1981-; Martin, Stephen F.A series of conformationally constrained and flexible pseudopeptides were prepared and their thermodynamic parameters on binding to the Grb2 SH2 domain were determined by isothermal titration calorimetry (ITC). Cyclopropane constrained analogs having hydrophobic amino acid residues at the pTyr+1 position exhibited, on average, a 2-5 fold improvement in binding affinities with the enhancement in affinities due to a more favorable enthalpy, not entropy, of binding. This serves as the first set of examples which demonstrate that favorable entropies of binding are not an inherent characteristic of ligand preorganization. Incorporation of polar amino acid residues at the pTyr+1 position eventuated in a slightly different thermodynamic effect than what was observed with the hydrophobic analogs. The constrained molecules exhibited greater binding affinities for the Grb2 SH2 domain and that increase in affinity was a consequence of a more favorable enthalpy, not entropy, of binding. However, the binding entropies for the polar set of constrained and flexible molecules were all negative. Structural information obtained from the co-crystallization of selected constrained and flexible ligand pairs with the Grb2 SH2 domain revealed an increase in conformational mobility of the BC loop in the complexes of the constrained derivatives and the presence of a greater number of direct polar contacts, but fewer water-mediated interactions between the phosphate group of the constrained molecules and the pTyr binding pocket of the domain. The construction of the cis-hydrindanone ring system in sieboldine A was accomplished utilizing either a Lewis acid-mediated silyl-directed Nazarov cyclization of a functionalized divinyl ketone or a sequential Ni(0)-catalyzed 1,4-addition/5-endo-dig cyclization. Propargylzinc bromide was shown to undergo conjugate addition to the [alpha]-aminopropyl substituted enone using Ni(acac)₂, thus providing a new, mild protocol for the conjugate propargylation reaction. Further efforts toward the formation of the α-epoxy ketone are described.Item Development and application of small molecule chaperones for protein renaturation(Texas Tech University, 2003-12) Liu, Xiangyi; Flowers II, Robert A.; Birney, David M.; Pare, PaulProtein aggregation presents a major problem in protein renaturation. This is due to competing intramolecular and intermolecular processes in unfolded protein molecules. Current investigations in our lab have focused on examining additives that can aid denatured polypeptide chains to refold into their native conformations and stabilize the structure of resulting proteins. Our approach to this problem is the development of a series of fluorous and non-fluorous salt additives that are capable of stabilizing protein structure against irreversible thermal denaturation, allowing protein molecules to refold into their native conformations, and recovering protein activities after chemical denaturation. Our initial studies employed Hen Egg White Lysozyme (HEWL) and Carbonic Anhydrase B (CAB) as model proteins to explore the effects of these salts on protein stabilities and renaturation. We also studied inclusion bodies of MMP 13 provided by Pfizer, Inc, and examined the utility of our protocol in a more practical application. The behavior of fluorous and non-fluorous salts prepared in our lab was investigated in the case of both thermal and chemical denaturation of HEWL and CAB. Differential Scanning Calorimetry (DSC) was employed to monitor the thermal process of protein solutions treated with salt additives. While many of those salts are able to prevent aggregation of HEWL during heating at a high temperature, fluorous salts were found to have an unusual stabilizing effect on protein structure. Moreover, compared with non-fluorous salts, fluorous salts could generate higher recovered enzymatic activities from chemically reduced-denatured HEWL at a useful concentration. The structure of recovered proteins was investigated further using Circular Dichroism (CD) spectroscopy. Although difficulties were encountered in attempting to prevent the irreversible thermal denaturation of CAB, fluorous salts have significantly enhanced the recovery of chemically denatured CAB molecules at a relatively high concentration. In our attempt to renature MMP 13 inclusion bodies, we obtained the results that further confirm the critical importance of the fluorous character in salt additives, hi addition, all the refolded-active proteins were separated from the salts by a simple dialysis protocol. Our approach to protein renaturation has a number of advantages. Furthermore, the observation of the important role of fluorous portion in stabilizing and recovering native protein structure provides the potential to develop a series of highly efficient additives for protein renaturation.Item Effects of processing on digestible amino acids of corn hybrids for ruminants(Texas Tech University, 1997-05) Holthaus, Dennis L.Three experiments were designed and conducted to determine the effects of steamflaking com hybrids on the digestibility of essential amino acids, for use in more exact formulation of diets for cattle. Information on the digestibility of amino acids by cattle is very limited and could see immediate industry application. A series of experiments were conducted to measure the digestibility of lysine, methionine, threonine, dry matter and nitrogen content of four com hybrids. In the first experiment, one-hundred-twenty weanling rats were utilized in a 4 x 3 factorial to determine the digestibilty of the limiting amino acids for cattle. The rats were randomly assigned to individual wire bottom cages and fed one of twelve experimental diets containing one of four unprocessed corn hybrids supplemented with the ten essential amino acids except for the amino acid of interest. After seven days adaptation and seven days collection in the first trial, rats were rerandomized into a second trial with the source of com being steam-flaked. Amino acid profiles were determined by using HPLC. A follow-up experiment was then conducted to determine the rumen degradation of the limiting amino acids for cattle utilizing in vitro techniques of both processed (steam-flaked) and unprocessed grains incubated for 4, 8, and 16 h. The final experiment was conducted to determine the total tract digestion of lysine, methionine and threonine of the same steam-flaked hybrids in steers using a 4 x 4 latin square design. Total collection of feed, feces, urine and orts was used to determine the amino acid digestion, nitrogen retention and dry matter digestion. Differences (P <.05) were found for all variables measured between the processed and unprocessed grain sources when fed to rats. Lysine and methionine digestibility was the highest for Hybrid 2 and was significantly higher (P < .05)than all other hybrids. Hybrid 4 had the highest threonine digestibility and was greater than (P < .05) remaining hybrids. Digestion of methionine was higher (P < .05) than lysine or threonine and performance of rats fed the methionine deficient diets was greater (P < .05) than remaining amino acids. Dry matter digestibility in vitro tended to be higher for the steam-flaked grain when compared to the unprocessed grain.Item Evaluating the technique of using nitrogen retention as a response criterion for amino acid studies in the horse(Texas A&M University, 2007-09-17) Antilley, Teri JillSix Quarter Horse yearling fillies were used in a duplicated 3 x 3 Latin square designed experiment to evaluate the technique of nitrogen retention as a response criterion for amino acid studies in the horse. The yearlings were paired by age and randomly assigned to one of three concentrates fed with a medium quality Coastal Bermudagrass hay throughout the study. Diets were fed at approximately 1.9% of horse body weight per day, divided into twice daily feedings with a 60:40 concentrate: hay ratio. With the exception of lysine and threonine, proposed amino acid requirements for yearling horses were calculated using nutrient to calorie ratios of gilts weighing 80-120 kg and gaining 325 g/d. Diet A was amino acid sufficient, as provided by a soybean meal-based concentrate. Diet B was amino acid deficient, with a cottonseed hull-based concentrate. Diet A and Diet B were isonitrogenous, containing approximately 12% crude protein. Diet C used the identical concentrate as Diet B, with synthetic essential amino acids and cysteine orally dosed to match the amino acid levels in Diet A. Nitrogen retention was not different between Diet A and Diet B. Diet C resulted in differences from Diets A and B in nitrogen retention; however, differences were a consequence of nitrogen intake. Nitrogen retained as a percent of nitrogen absorbed was lower (P < 0.05) for Diet B than for Diet A, for data not accounting for endogenous fecal and urinary losses. There were no differences in nitrogen retained as a percent of nitrogen absorbed for horses fed Diet C, when compared to either Diet A or Diet B, for data not accounting for endogenous losses. It was concluded that differences in nitrogen retained as a percent of nitrogen absorbed were observed between amino acid sufficient diets and amino acid deficient diets. However, horses fed amino acid deficient diets and orally dosed with synthetic amino acids, likely require some modified dosage level to achieve the same or higher values in nitrogen retained as a percent of nitrogen absorbed as those values for amino acid sufficient diets.Item Exploration of adaptation to unnatural amino acids(2002-08) Bacher, Jamie Mitchell; Ellington, Andrew D.Item Genotypic variability in protein content and amino acid composition of pearl millet (Pennisetum Typhoideum)(Texas Tech University, 1978-05) Han, Ruth Tan-PiNot availableItem Investigation of immobilized biopolymers for metal binding(2004) Malachowski, Lisa Lyn; Holcombe, James A.This research focuses on the utility of immobilized poly amino acids for metal remediation and preconcentration. The biohomopolymer poly-L-histidine (PLHis) was immobilized onto controlled pore glass (CPG) and its metal binding capabilities evaluated through the use of a flow injection analysis - flame atomic absorption system (FIA-FAAS). The metal binding capability of PLHis-CPG was determined through the analysis of the generated breakthrough curves. The polymer likely coordinates cationic metals through the imidazole side chain (pKa ≈ 6) present on each histidine residue with both strong and weak binding sites for Cu2+, Cd2+, Co2+, and Ni2+. It has also been shown that the protonated imidazole side chain present in acidic conditions is capable of binding metal oxyanions such as chromates, arsenates, and selenites; although oxyanion binding currently exhibits interferences from competing anions in solution, such as sulfate and nitrate. Poly-L-Aspartic Acid (PLAsp) and Poly-L-Glutamic Acid (PLGlu) were also individually immobilized onto controlled pore glass (CPG) and compared using their metal binding capabilities. Elemental combustion analysis was used to yield polymer coverage approximations. Formation constants and site capacities of both polymers for Cd2+ were determined through equilibrium and breakthrough studies. Additionally, the metal selectivity of PLAsp and PLGlu was evaluated when breakthrough curves were run with several metals present in solution at one time. Both polymers exhibited similar binding trends and binding strengths for all of the metals studied. This likely reflects the absence of a predetermined tertiary structure of the polymers on the surface and the relatively high residue-per-metal ratio (~20:1), which places less stringent requirements on the steric hindrance between the side chains and the resultant ìwrappingî of the peptide around the metal. Initial attempts at determining formation constants of PLAsp and PLGlu through competitive binding experiments with either EDTA or oxalate present were unsuccessful due to complications caused by the current immobilization procedure. Therefore, alternate immobilization procedures were investigated utilizing an epoxide linker. These methods eliminate the formation of an amine functionality on the surface. Additionally, a combinatorial approach was used in an attempt to elucidate an optimal copolymer primary structure for successful binding of a target metal. This approach included screening the library for successful binding with micro x-ray fluorescence (MXRF) and obtaining the sequence of the successful copolymer through Edman Degradation. A considerable amount of the metal binding experiments conducted in this research used the analysis of breakthrough curves generated through flow injection-flame atomic absorption spectrometry. Solution flow rate is a critical parameter in breakthrough analysis. Due to the absence of an inexpensive, on-line flow meter for flow injection analysis systems, an electronic flow meter was constructed to measure the flow rate during the FIAAS measurements. Thus, flow rates can be measured while collecting breakthrough data, and continuous monitoring of flow rates is possible.Item Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids(Texas Tech University, 1985-05) Anderson, Clinton HNot availableItem Model systems for the 330 nm chromophore of pyridoxal 5'- phosphate containing enzymes(Texas Tech University, 1976-08) Kang, Mohinder SinghThe absorption, fluorescence and chemical properties of the PLP site in enzymes can be simulated by model systems (9). The emphasis of this dissertation is the development of model systems for the three structures; carbinolamine, non-ionic hydrogen bonded Schiff base and substituted aldimine, proposed for the 330-335 nm chromophore of PLP containing enzymes. The PLI adducts with di- and triethylam ine were studied as models for the carbinolamine structure; the PLP adducts with t-butylarine and n-hexylamine were shown to be models for the Schiff base; and the adducts between diaminopropane and pyridine-4-aldehyde and benzaldehyde were studied as a model of the substituted aldimines structure.Chemical and physical properties of these model systems are presented and discussed in relation to PLP adducts with enzymes. Absorbance, infrared, fluorescence and NMR spectra were used to characterize the adducts. The effects of different solvents (DMSO and H„0) on PLP-amine adducts are reported and discussed.Item Mutagenesis of cyclic AMP receptor protein: targeting positions 72 and 82 of the cyclic nucleotide binding pocket(Texas Tech University, 1992-12) Belduz, Ali AsmanThe cyclic adenosine 3',5' monophosphate (cAMP): cAMP receptor protein (CRP) complex functions to promote transcription activation of several operons in Escherichia coli. The complex binds to specific DNA sites located upstream of, for example, the lactose operon promoter (lacP). This activates lacP and allows high level expression of lactose operon structural gene sequences. Five specific amino acid residues are predicted to play a role in the interaction of cAMP with CRP. The purpose of this work was to test the role of two amino acid residues, glutamate at position 72 and arginine at position 82, in facilitating both cAMP binding and cAMP-mediated activation of CRP. Mutants at positions 072 and 082 in the cyclic nucleotide binding pocket of the CRP were constructed by site directed mutagenesis. Glutamate at position 072 was substituted by either leucine, glutamine, or aspartate. Arginine at the position 082 was substituted by either lysine, histidine, leucine, isoleucine, or glutamine. A parallel set of substitutions was constructed in a CRP*, or cAMP-independent, form of the protein to determine the effects of the amino acid substitutions on CRP conformation. Each mutant form of CRP was characterized in vivo with respect to sugar utilization, and through quantitative pgalactosidase activity and in vitro by measurement of cAMP binding activity under near physiological salt concentrations. The results of this study confirm a role for both glutamate at position 72 and arginine at position 82 in cAMP binding and activation of CRP; neither aspartate at position 72 nor lysine at position 82 could effectively substitute for glutamate or arginine in an otherwise wild-type CRP. The results of this study also indicate that cGMP, a competitive inhibitor of cAMP binding to CRP, binds the cyclic nucleotide binding pocket differendy than cAMP.Item Permeabilities to low molecular weight solutes of collagen and regenerated cellulose dialysis membranes.(Texas Tech University, 1974-08) Dearden, Craig LeeNot availableItem Phosphorylation of hexokinase by tyrosine and serine/threonine specific kinases(Texas Tech University, 1992-12) Seale, Jeffrey WadeNot availableItem Protein engineering on soybean sterol methyl transferase leads to altered substrate binding and catalysis(Texas Tech University, 2004-12) Sinha, ArchanaSterol methyltransferases (SMTs) are ubiquitously represented in plants and they can serve as the rate-limiting enzymes in the 24-alkyl sterol (phytosterol) pathway. Together these enzymes are capable of converting sterol acceptors with a 24(25)-double bond (cycloartenol, CA; CI-activity) or 24(28)-double bond ((24)28-methyleneIophenol, ML; -C2-activity) in the sterol side chain into more that 60 distinct phytosterols in a single plant. Recently, we discovered using the soybean SMT that depending on the nature of the substrate olefin bound to SMT, either one or two catalytic reactions can proceed, concerted or step-wise, to generate the product diversity. To investigate the proposed role of aromatic amino acids that are part of a signature motif in the active site of SMT enzymes- F82YEYGWG88, Y83L and Y83F mutants were prepared and purified to homogeneity and the steady-state kinetic parameters were determined as described in this laboratory for the wild-type soybean SMT. The Y83L mutant performed much like the wild-type enzyme in terms of substrate acceptability, product distribution and physical property, but differences were detected in Y83F mutant. "When the mutant SMT activities were compared to the native SMT activities in relation to inhibition by 25- azacycloartenol (transition state analog) or 26,27-dehydrocycloartenol (mechanism based inhibitor), both sets of enzymes were found to be inhibited with equal efficacy, suggesting that the successive C-methylation of the Ä24 bond occurs at the same active center. Based on activity assays performed over the temperature range 15 to 40''C, the activation energy (Eact in KJ/mol) estimated from the Arrhenius plots were found to be: (i) wild-type, CA = 49, ML = 71; (ii) Y83F, CA = 65, ML = 52 and (iii) Y83L, CA = 98, ML =185. Analysis of the pH dependence of log kcat/Km for the wild-type and two mutants showed different profiles for Ä 24(25) and Ä24(28) -substrates. The results of the mutational and kinetic analyses are interpreted to imply that product diversity catalyzed by the soybean SMT is made possible by the relaxed control over substrate and intermediate conformations resulting from altered cation-ð interactions in the active sites of the mutant enzyme and relates to the different mechanisms catalyzed using different olefin substrates.