Biochemistry, Biophysics, and Structural Biology Commons^™

The Involvement And Relationship Of Ferritin And Ceruloplasmin In Oxygen Radical Toxicities, David Wilson Reif

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The intent of this dissertation is to demonstrate that ferritin iron may be involved in alloxan, diquat, and radiation-dependent damage and that ceruloplasmin may afford protection by limiting the availability of ferritin iron. Initial studies demonstrated that the alloxan radical formed by glutathione-dependent reduction of alloxan reductively mobilized ferritin iron and the released iron catalyzed lipid peroxidation. SOD and catalase were without effect, whereas ceruloplasmin effectively inhibited both iron release and lipid peroxidation. Using ¹³⁷Cs gamma-radiation it was demonstrated that radiolytically generated superoxide released iron from ferritin with an efficiency of 67%. Lipid peroxidation strictly required both superoxide and …

Kinetics Of Two-Substrate Reactions Using Integrated Rate Equations, Leonard D. Holmes

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The usual procedure employed in enzyme kinetic analysis is the method of initial rates. However, it has been appreciated for years that the analysis of enzyme-catalyzed reactions could, in principle, be more efficiently performed by examining the entire time course. There is much more information contained in a progress curve than in a simple initial rate. With the appearance of the computer, the formidable computations necessary for the use of integrated-rate equations are quite possible. The intention of this research was to develop the analytical and statistical methodology for applying an integrated-rate equation to a two-substrate reaction. I have analyzed …

Mechanism Of Iron Transport In Mycelia Sterilia Ep-76, Jonathan P. Adjimani

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The cyclic trihydroxamic acid, N, N', N' '-triacetylfusarinine C, produced by Mycelia sterilia EP-76, is shown to be a ferric ionophore for this organism. The association constant for ferric-N, N', N' '-triacetylfusarinine C complex was determined to be log K=32.5. Other iron chelating agents, such as rhodotorulic acid, citric acid, or the monomeric subunit of triacetylfusarinine C, N-acetyl- fusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating …

An Improved Method For Transferring Nucleic Acids To Nylon Membranes, Bruce D. Parker

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Methods currently in use for the separation and identification of specific segments of nucleic acids involve long transfer periods or elaborate apparatuses and result in the production of a single blot. Contamination by organisms or enzymes is always a factor to be dealt with. An improved method for transferring nucleic acids from acrylamide or agarose gels for use in hybridization has been developed. This method uses NaOH as the blotting medium to improve the rate and efficiency of transfer to nylon membranes. As many as six blots can be obtained within one hour using this method. This method is effective …

Time Course Of Enzyme Catalyzed Reactions: The Stoichiometry A --> P + Q, Tamra Tuttle

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The feasibility of using an integrated rate equation to analyze the kinetics of a second-order, enzyme-catalyzed reaction has been investigated. The inducible arginine decarboxylase from Escherichia coli B. was chosen for this study because it catalyzes an irreversible reaction with stoichiometry A --> P + Q, the simplest second order case. Values for five of the eight possible kinetic constants were determined from 21 time courses. Of the remaining three, the uncompetitive product inhibition constant for CO₂ was shown to be between 0 and 0.06, while the values of the competitive product inhibition for CO₂ and the uncompetitive …

Photoaffinity Labeling Of The Antimycin Binding Site In Rhodopseudomonas Sphaeroides, Emily Wilson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The purpose of this study was to identify the site of interaction of antimycin with the ubiquinone-cytochrome b-c1 oxidoreductase in the photosynthetic bacteria, Rhodopseudomonas sphaeroides. To accomplish this goal, three areas of research were undertaken: the synthesis of a radiolabeled, photoaffinity analog of antimycin, identification of the inhibitory characteristics of this analog, and the photoaffinity labeling of the antimycin binding site. All three areas were accomplished.

The major finding of this study was the identification of an 11,000 dalton polypeptide as the predominantly labeled protein. Although this polypeptide was not exclusively labeled, it was consistently labeled and showed competition …

A Study Of The Action Of Bovine Cathepsin D On Intramuscular Connective Tissue, Elimosaria Elikalia Maeda

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Cathepsin D has been postulated to play a role in the tenderization of meat with ageing. Meat toughness has been related to connective tissue content and degree of myofibrillar shortening. This investigation was undertaken to determine the activity of cathepsin D on bovine intramuscular connective tissue.

Highly purified cathepsin D preparations from bovine spleen and bovine sternomandibularis muscle were obtained by ammonium sulfate fractionation followed by affinity chromatography on pepstatin coupled to controlled pore size alkyl amine glass beads. Maximum proteolytic activity using hemoglobin as substrate was obtained at pH 3.5 and 3.8 for bovine spleen and muscle cathepsin D, …

Hemoglobins Of The Cutthroat Trout Salmo Clarki, Jonathan N. Southard

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Nine hemoglobins have been isolated from the blood of cutthroat trout. All nine hemoglobins bind oxygen cooperatively and appear to be tetramers with molecular weights of -64,000. The oxygen equilibria and subunit structures of the purified hemoglobins were studied. In addition, the red blood cells of cutthroat trout were examined for the presence of ATP and GTP, which are known to be physiological modulators of hemoglobins in fishes.

Five hemoglobins with isoelectric points from 9.1 to 7.0 are classified as cathodal hemoglobins. These five hemoglobins have identical oxygen binding properties by the criteria tested. All have oxygen equilibria which are …

Subunit Interactions In The Inducible Arginine Decarboxylase From Escherichia Coli B, Catalina N. Depusoy

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The nature of the subunit interactions in the inducible arginine decarboxylase from Escherichia coli B is of considerable interest because of the observed differences in the catalytic activities of the dimer and the decamer; the decamer is active and the dimer is inactive. To study these interactions, inactive dimers were prepared by sodium borohydride reduction of the E-amino--pyridoxal-P Schiff base. Hybrid decamers were then prepared from varying molar ratios of native and reduced dimers. The hybrid decamers were indistinguishable from native decamers as observed in the analytical ultracentrifuge and on acrylamide gel electrophoresis. Kinetic studies indicated that true hybrids were …

The Isolation And Characterization Of A Novel Iron Protein From Desulfovibrio Gigas, Alan Kay Smith

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. This protein was treated with acid, chelating agents, detergents and proteases in order to study their effect on the iron solubility. The results of elemental analysis of a crude protein preparation were 5.3% iron, 2.9% sulfur and 11 .9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be …

Assay, Purification, And Characterization Of A Pantetheine Hydrolyzing Enzyme From Pig Kidney, Carl Thomas Wittwer

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A microsomal glycoprotein hydrolyzing pantetheine to pantothenate and cysteamine has been solubilized and purified to homogeneity as determined by sodium dodecylsulfate electrophoresis. Four rapid, independent assays of pantetheine hydrolysis are described and compared along with a method for localizing enzymatic activity on polyacrylamide gels. The enzyme is solubilized on exposure to butanol and purified by heat treatment, (NH4)₂SO₄ fractionation, hydrophobic chromatography, and hydroxyapatite chromatography. The glycoprotein, purified 5600-fold in 22% yield, has a specific activity of 14 μmoles pantothenate produced/min/mg of protein, 35 times that previously reported. The enzyme has a pH optimum of 9.0-9.5 and a …

The Transport And Intracellular Distribution Of Iron In The Basidiomycete, Ustilago Sphaerogena, Charles W. Passavant

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The transport and intracellular distribution of iron in Ustilago sphaerogena via the native siderochrome ferrichrome A has been examined. Evidence is presented which suggests the involvement of deferriferrichrome in iron transport when the metal is supplied to the cell as ferrichrome A. This process requires both oxygen and ATP synthesis and may require a transmembrane electrochemical gradient. Once inside the cell, iron derived from ferrichrome A is rapidly distributed, with a major fraction found in the mitochondria, presumably as heme.

The kinetics of ferrichrome and ferrichrome A uptake have also been investigated. The Km of transport of ferrichrome was …

An Enzyme-Linked Immunosorbent Assay (Elisa) For Pantothenate, Allen H. Smith

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

An enzyme-linked immunosorbent assay (ELISA) for pantothenate has been developed. Antibodies induced in rabbits against bovine serum albumin-pantothenate conjugate were specifically purified by affinity chromatography. This process served to reduce the amount of endogenous pantothenate attached to the antibody, as well as to purify the antibody. The purified antibodies were covalently linked to alkaline phosphatase (Sigma type VII) with glutaraldehyde (0.05% aqueous solution). An immobilized pantothenate substrate was first obtained by attaching human serum albumin-pantothenate conjugate to the surface of polystyrene culture tubes by passive adsorption. The binding of the enzyme labelled antibody (E-AB) to this substrate is proportionately inhibited …

Cow Brain Glutaminase: Purification, Characterization, And Mechanism Of Action, Fulung John Chiu

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A simple, quick, and sensitive radiometric assay for glutaminase has been established. This assay uses L-(U-¹⁴C)-glutamine as the substrate and measures the ¹⁴CO₂ produced when the reaction is coupled to glutamate decarboxylase. An efficient purification procedure for cow brain glutaminase has also been developed; the yield is 12%. Steps include acetone extraction, ammonium sulfate fractionation, calcium phosphate gel treatment, and differential gel filtration on Sepharose 4B. The final preparation has a specific activity of 385 μmole/min/mg and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate; this band corresponds to a …

A Unified Radiometric Assay System For The Gaba-Glutamate Regulating Enzymes, Robert C. Dinwoodie

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The purpose of this paper was to develop a single assay system for the enzymes which regulate GABA and glutamate concentrations in brain and nerve tissue. Since all the enzymes produce L-glutamate, their activities were measured by coupling them to L-glutamate decarboxylase. Enzymatic activity was determined by measuring the release of co2 from radioactive substrates. The glutamate decarboxylase was obtained from a commercial acetone powder by simplifying existing procedures. The glutamate decarboxylase produced was of sufficient purity to be used in the coupled assays, which were checked with commercial preparations of each enzyme, where available, and with crude brain homogenates. …

Model Studies For Molybdenum Enzymes. The Reduction Of Cytochrome C By Molybdenum(V)-Cysteine Complexes, Glen D. Lawrence

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The reduction of ferricytochrome c ( cyt c^III), as well as some iron ligand complexes and chemically modified derivatives of cyt c^III, by two molybdenum(V)-cysteine complexes has been investigated as a model for electron transfer in molybdenum enzymes. The reduction of cyt c^III by di-μ-oxo-bis [oxo(L-cysteinato)molybdate(V)] (I) is first order in cyt c^III and zero order in I over a wide range in concentration of I at neutral pH. The reduction of cyt c^III by μ-oxo-bisfoxodihydroxo(L-cysteinato) molybdate(V)] (II) is first order in both reactants and has a rate several orders of magnitude greater …

Model For Estimating Soil Water Flow, Water Content, Evapotranspiration And Root Extraction, Musa N. Nimah

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A mathematical model was developed to predict water content profiles, evapotranspiration, water flow from or to the water table, root extraction and root water potential at the surface as functions of time under unsteady state conditions.

The model was tested in the field at The Hullinger Farm near Vernal, Utah, in 1970 and 1971. Comparison of water content-depth profiles show excellent agreement at the end of a 9- day run in 1970 on oats seeded to alfalfa. In 1971 with alfalfa as the crop, the data show best agreement, between predicted and computed water content-depth profiles, 48 hours after any …

The Regulatory Effects Of Certain S-Triazine Compounds On The Nutritive Composition Of Seeds And Leaves Of The Pea (Pisum Sativum L.) And Sweet Corn (Zea Mays L.) And Responses Of Metabolic Systems, Ming-Tsung Wu

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The study was conducted to investigate the influence of eight s-triazine compounds--simazine, atrazine, terbutryne, GS-14254, propazine, prometryne, prometone, and ametryne--on the composition of seeds of peas and sweet corn through soil and foliar application. In order to understand the mechanism of the effects of these a-triazines on protein accumulation, the responses of the metabolic systems of pea and sweet corn seedlings to s-triazine treatment was studied with emphasis on the stimulation of certain enzymes, and the changes in the rates of incorporation of ¹⁴C-L-leucine (U) into soluble protein. In addition, the effect of simazine on the ultrastructure …

The Effects Of Sources And Levels Of Vitamin A And D On Some Aspects Of Sulfur Metabolism, Akbar Malekpour

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Two trials were conducted using 48 infant pigs of both sexes. Pigs were taken from the sow when three to seven days old and placed in wire-bottomed metal cages by pairs. Animals in the first trial received synthetic diets containing 100 or 1000 international units of ergocalciferol and 1000 or 4000 international units of retinyl acetate or four or sixteen milligrams of beta carotene per kilogram of feed. Baby pigs in the second trial received the same synthetic diet plus 1000 international units of ergocalciferol (D₂) or cholecalciferol (D₃) and 2000 international units of vitamin A …

Gas Chromatographic Determination Of Changes In Concentration Of Pipecolic Acid And Other Free Amino Acids In Hypersensitive Tobacco Plants Induced By Tobacco Mosaic Virus And Temperature, David Francis Brown

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Changes in concentrations of pipecolic acid and other free amino acids in Nicotiana tabacum, L. cv. Xanthi induced by tobacco mosaic virus (TMV) incubated at 75 or 95 F after inoculation were investigated gas chromatographically. A preliminary extraction and gas chromatographic technique for separation and quantitative determination of the free amino acids, using hydroxyproline as an internal standard, was developed.

Amino acids were converted into their trifluoroacetyl n-butyl (TAB) derivatives and gas chromatographed on 0.325 percent ethylene glycol adipate (EGA) columns. Resolution was generally good, but glycine-isoleucine and phenylalanine-aspartic acids were not resolved on old columns. The over-all …

Quantitative Evaluation Of Saponin Content In Du Puits Alfalfa Foliage (Medicago Sativa L.) With Trichoderma, Wang Li-Chun

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Selections in Du Puits alfalfa (Medicago sativa L. ) were evaluated for saponin content. The data obtained from the Trichoderma bioassay were converted to saponin values by regressions and standard curves. It was necessary to adjust the concentration of alfalfa extract in the bioassay so that the saponin content in alfalfa was estimated in that portion of the curve showing a marked response to saponin. A different regression was established for each group of low and high saponin plants of Du Puits alfalfa. The standard curve prepared with extract of Du Puits alfalfa used on the basis of its …

Biosynthesis Of Piperidine-2-Carboxylic Acid In Fluoride-Necrotic Glycine Max, Susan S. Martin

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Synthesis of piperidine-2-carboxylic acid (pipecolic acid) in soybean was studied by infiltration of radioactive carbon labeled precursor and by experiments using enzyme extracts from control and fluoride-fumigated plants.

Pipecolic acid was synthesized from lysine in both control and treated plants. Normal plants, in which pipecolic acid had not been detected by conventional procedures, were capable of synthesizing pipecolic acid. Greater pipecolic acid synthesis was shown in treated plants than in controls, and was attributed to increased enzyme concentration induced by fluoride treatment.

The Biosynthesis Of Aminolevulinic Acid In Relation To Iron And Enzyme, Daniel Ming-Yi Teng

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The survey of plant species for aminolevalinic acid synthetase (ALAS) source was conducted. Under the conditions of this study, spinach leaves were found to be a good source of ALAS activity. The amount of amino levulinic acid (ALA) synthesized by the plant extracts was very small and was rapidly metabolized to porphyrins. ALA and porphyrin synthesis was found to be confined to the particulate fractions. Rupturing of the particles with ultrasonifier enhanced the penetration of succinyl-CoA and increased the activity. Direct study of the ALAS activity was enabled by the utilization of C¹⁴-compounds to the isolation by column …

Interaction Of Inorganic And Organic Fluoride Ions With Available Calcium On Root Growth In Triticum Vulgare, L., Subbanaidu Ramagopal

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A method has been described to suspend seedling wheat roots in a saturated atmosphere. Wheat (Triticum vulgate, L.) seeds were germinated in aluminum trays and the seedlings transferred to seedling chambers. After a day's growth, the roots were treated with various concentrations of solutions containing CaSO₄, KCl, KF, NaCL, NaF, CH₃COONa, FCH₂COONa, K₂C₂O₄ and Na₂EDTA and observations were recorded 24 and 48 hours after treatment. The new root growth was described with respect to root hairs, root tissue collapse and root discoloration. Total length and …

Determination Of Estrone, 17b-Estradiol And Estriol In Urine Of Young Adults, Shiao-Fan Lee

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A study was made of the urinary excretion of estrogens of healthy university students consuming self-selected diets living under home conditions (4 men and 5 women). Two 31-day test periods were used. During the two test periods, two meal patterns were used; three meals a day or two meals a day with no lunch. Weight was kept constant. Calories, protein, carbohydrate, and fat of their diets were calculated.

Urine specimens were collected twice a week, and additional samples were also collected on specified days for female subjects. Urinary estrogens were determined by using Brown's method (1955, 1957) with some modifications. …

Fluoroorganic Compounds In Relation To Hydrogen Fluoride Exposures In Leaves Of Glycine Max Merr., Julie Ye-O Cheng

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Soybean plants were fumigated in HF and the organic acid-fractions of the leaf extracts were compared to those of the leaf extracts from soybean plants grown in nutrient solution containing fluoroacetate. The extracts from both treatments contained fluoroorganic compounds that were chromatographically similar to fluoroacetate and fluorocitrate.

The fluorocitrate separated from HF fumigated leaves was found to inhibit aconitase prepared from pig-heart. This inhibition was similar to that caused by commercial fluorocitrate and the fluorocitrate synthesized using guinea-pig kidney. The aconitase from soybean leaves was found to be sensitive to fluorocitrate as the aconitase that isolated from pig-heart. Inorganic fluoride …

Alkali Liberated Phenolic Acids From Alfalfa, Leslie Huey-Seng Huang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Fourteen compounds were isolated from an alkaline hydrolyzed extract of alfalfa leaves by using cellulose thin layer plates. The R_f values, fluorescent characteristics, and color reactions were given for all the compounds isolated.

Eight of these were identified as trans-ferulic acid, trans-p-coumaric acid, trans-caffeic acid, o-coumaric acid, tricin, syringic acid, cis-ferulic acid and cis-p-coumaric acid. The identity of the compounds was confirmed by comparison of the R_f values on thin layer chromatograms, fluorescent characteristics under ultraviolet light, and color reactions of spray reagents with those of authentic samples. Three of …

The Complex Formation Of Silver Ion With Ribonucleic Acid, Guanosine, Inosine And Related Compounds And Peroxidase-Like Activity Of A Haemundecapeptide Prepared From Horse Heart Cytochrome C, José Angel Reinosa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The importance of nucleic acids in plant and animal cells as carriers of genetic information and as protein biosynthesis agents is well recognized. It is also known that nucleic acid is a component of all viruses.

Takahashi (45) and Fraenkel-Conrat (16) demonstrated that the protein component of tobacco mosaic virus is non-infectious to the host plant, although it is identical to the original virus morphologically. The virus ribonucleic acid (RNA) alone was infectious, however.

Deoxyribonucleic acid (DNA), which is present in chromosomes, displays a very specific function. The chromosome long has been accepted as the carrier of the hereditary unit, …

Saponin Content And Some Pod And Blossom Characteristics Of Alfalfa As Related To Seed Infestation By The Alfalfa Seed Chalcid, Ronald D. Morse

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The alfalfa seed chalcid, Bruchophagus ruddi Guss., is a jet-black hymenopteran wasp. The destructive nature of this pest has been recognized since the latter part of the nineteenth century. Every year thousands of acres of alfalfa seed are destroyed, with infestation reaching as high as 8 5 percent in some areas. In Utah the chalcid annually ruins from 5 to 25 percent of the alfalfa seed. Much of this damage goes unnoticed, as infested seed is commonly blown out in the trash during harvesting and cleaning operations . The extent of damage is not restricted to the United States. Wherever …

The Effect Of Fluoride On C14O2-Fixation And The Synthesis Of Organic Acids, Amino Acids, Protein And Carbohydrates, Chin-Fung Li

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Many studies have been conducted on the effect of fluoride on plants, but the exact mechanisms by which fluoride causes damage is little understood. The following physiological and metabolic processes are known to be affected by fluoride: a decrease in chlorophyll (McNulty and Newman, 1961), a diminished rate of photosynthesis (Thomas and Hendricks, 1956; Bishop, et al., 1958), decreased plant growth (Bonner and Thimann, 1950), inhibition of ascorbic acid synthesis (Smith, 1952), variation in starch and total polysaccharides (Donald and Emerson, 1961), inhibition of glucose transport (Jiri, et al., 1962), inhibition of metabolism (Ordin and Skoe, 1963), effects …