Biochemistry Commons^™

Aspects Of Mitochondrial Oxidative Metabolism, Leslie Kushner

Dissertations, Theses, and Capstone Projects

The effects of metabolic intermediates and coenzymes on the activities of fatty acyl-CoA synthetase and carnitine palmitoyltransferase (CPT) in rat heart mitochondria were studied. ADP and palmitoyl-l-carnitine were weak inhibitors of fatty acyl-CoA synthetase. CPT A was inhibited 59.3% by 100 (mu)M succinyl-CoA. However, succinate thiokinase activity in rat heart cytosol was too low to maintain a succinyl-CoA concentration sufficient to affect the activity of CPT A. There was no indication that fatty acid oxidation in rat heart mitochondria is controlled via the regulation of either CPT A or fatty acyl-CoA synthetase.

The dependence of the oxidation of unsaturated fatty …

Correlation Studies Between Human Seminal Plasma Trace Metal Concentrations And Semen Quality, De-Shen Mei

Chemistry & Biochemistry Theses & Dissertations

Methodologies for the determination of cadmium, copper, selenium and zinc concentration in human seminal plasma have been developed using polarized Zeeman-effect atomic absorption spectroscopy. Within-run coefficients of variation (CY's) for cadmium, copper, selenium and zinc from pooled seminal plasma were found to be 8%, 5% , 4% and 0.5% at a mean (SD) concentration of 0.48 (0.04), 147 (7 .6), 53 (2. 1) and 122,900 (700) μg/ℓ, (n = 10), respectively.

Between-run CV's were determined to be 4% , 7%, 5% and 7% at a mean (SD) concentration of 2.4 (0.1), 147 (10. 1), 67.7 (3.4) and 115,800 (8300) …

Negative Cooperatively In The Binding Of 3-Aminopyridine Adenine Dinucleotide To L-Α-Glycero-3-Phosphate Dehydrogenase, David Wheeler

Chemistry & Biochemistry Theses & Dissertations

Glycerol-3-phosphate dehydrogenase catalyzes the reversible reaction:

NAD⁺ + G-3-P ⇌ NADH + DHAP + H⁺

The enzyme is found in organisms from E. coli to man. The primary function of the enzyme varies from organism to organism. Depending on the metabolism of the organism, the enzyme may function primarily to generate G-3-P for lipid (in organisms with a highly aerobic biosynthesis metabolism), or it may function primarily to produce NAD⁺ to support glycolysis. Complex multicellular organisms possess tissue specific isozymes of GPDH whose kinetic parameters reflect the relative importance of G-3-P and NAD⁺ production in various …

1-0-Alkyl-2-Acetyl-Sn-Glycero-3-Phosphorylcholine (Platelet Activating Factor): Interactions With Sheep Platelets, Duane O. Beaumont

Loma Linda University Electronic Theses, Dissertations & Projects

Platelet Activating Factor (PAF) seems to link platelet function with allergy and inflammation. Early events associated with the action of PAF on platelets are poorly understood, but PAF appears to interact with a platelet membrane receptor. Two photoaffinity-labelling analogs of PAF, diazoacetyl PAF and trifluorodiazoacetyl PAF, were designed for examining the interaction between PAF and its platelet receptor. These photoactive compounds were selected on the basis of the structure-activity relationship of PAF, and the general guidelines of the photoaffinity labelling technique. Some progress has been made in their synthesis and characterization.

The standard platelet aggregation assay was unreliable in testing …

The Effects Of Tri-O-Cresyl Phosphate On 2',3'-Cyclicnucleotide-3'-Phosphohydrolase Activity In Neural And Non-Neural Tissue Of The Rat, Mark K. Schlager

Chemistry & Biochemistry Theses & Dissertations

A total of 44 Long-Evans rats received 5.7 g/kg tri-O-cresyl phosphate (TOCP) orally every 3 days for 6 days. A second group of 13 rats received 4.6 g/kg orally every 3 days for 6 days. Animals were administered atropine sulfate (10 mg/kg) intraperitoneally every 6 hours or as needed to alleviate cholinergic symptoms. Ten control animals were given corn oil orally. Eight rats (18%) survived the 5.7 g/kg dose regimen and 5 rats (38%) survived the 4.6 g/kg dose regimen. Surviving rats were killed at 30-33 days post-dose. Signs of delayed neuropathy included ataxia and motor impairment.

The CNP activity …

Two-Dimensional Electrophoretic Studies Of Normal Spematozoal Proteins, Ying Lin

Chemistry & Biochemistry Theses & Dissertations

Protein profiles of human spermatozoa have been mapped by using high resolution two-dimensional electrophoresis which combines isoelectric focusing as the first dimension and polyacrylamide gradient gels electrophoresis in SDS as the second dimension. The profiles of normal spermatozoa! extraction, acrosomal extraction and seminal plasma showed high resolution in its separation capability. No qualitative or quantitative differences were observed by using Coomassie Brilliant Blue R-250 staining in a much higher concentration of proteins. However, coomassie blue lacks the sensitivity to detect many minor components. Therefore, a silver staining procedure was used, which resulted in greatly increased sensitivity.

Samples from normal fertile …

A Method For The Comparison Of Hdl Containing Apoprotein E And Hdl Cholesterol, Steven A. Knizner

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoproteins (HDL) were isolated by preparative salt density gradient ultracentrifugation and analyzed further by heparin-sepharose affinity chromatography. Separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation on a salt density gradient at 272,000 x g for 36 hours in a swinging bucket rotor. After isolation, the total HDL was subdivided into HDL containing apoprotein E and HDL without this apoprotein by heparin-sepharose affinity chromatography. Relative amounts of these subclasses were then calculated.

HDL-cholesterol was measured after precipitation of LDL and VLDL by phosphotungstic acid and magnesium. The statistical relationship between HDL …

Investigation Of The Coenzyme Binding Site Of Lactate Dehydrogenase From Chicken Muscle, Jia-Hwei Lin

Chemistry & Biochemistry Theses & Dissertations

Investigation of the coenzyme binding site of lactate dehydrogenase was approached by using coenzyme analogs. Inhibition studies of this enzyme using n-alkylphosphates as coenzyme analogs were performed with a fluorometric method. The results showed that all of the n-alkylphosphates were competitive inhibitors with respect to NAD⁺.

The n-alkyl-ADPs were synthesized from AMP and dicycloheaylammonium salts of n-alkylphosphates. The synthetic compounds were subjected to NMR spectroscopic studies, thin layer chromatography and determination of the ratio of adenosine moiety to phosphate groups. The results indicated that the synthetic compounds were n-alkyl-ADPs.

These synthetic compounds were used as coenzyme analogs in …

Comparison Of Surface Polysaccharides Of R. Leguminosarum Anu57 (Exo-1), R. Leg. Anu57(Pbr1an) (Nod+, Nif-), And R. Leg. Anu57(Pjb5j1) (Nod+, Nif-), Frank G. Hustmyer

Masters Theses

Extracellular polysaccharides (EPS) and lipopolysaccharides (LPS) are suspected to be involved in the legume/Rhizobium symbiotic process. Polysaccharide compositions from the EPS, LPS, capsular polysaccharide (CPS), and small polysaccharide (SmPS) were compared between Rhizobium leguminosarum ANU57 (parent, Exo^-1) and two mutants R. leg. ANU57(pBR1AN) (nod⁺, nif^-) and R. leg. ANU57(pJB5J1) (nod⁺, nif^-) both containing a Sym (symbiotic) plasmid insertion. R. leg. ANU57(pBR1AN) and R. leg. ANU57(pJB5J1) are nod⁺ on clover and peas, respectively. The nod⁺ mutants produce larger amounts of EPS and decreased amounts of LPS compared to …

Synthesis And Characterization Of Some Oxoperoxopolycarboxylato Vanadates (V) And Their Biochemical Significance, Myunghi Lee

Dissertations, Theses, and Masters Projects

No abstract provided.

Evaluation Of Peptide Assimilation In Ehrlich Ascites Tumor Cells, John M. Castellano

Biological Sciences Theses & Dissertations

The dipeptides glycyl-L-(³H)-leucine and L-phenylalanyl-L-(³H)-leucine were synthesized in radioactive form and utilized in the analysis of peptide transport and hydrolysis by Ehrlich ascites tumor cells. The tumor cells were cultured in the abdominal cavity of CF-1 albino mice and harvested into sterile isotonic buffer for all experimental analyses.

The results of this study reveal that the tumor cells rapidly accumulate radiolabel when incubated with the above mentioned peptides, but that the peptide is most probably hydrolyzed extracellularly and free L-(³)-leucine transported. This hypothesis is based on the observations of reduction in accumulation of radiolabel …

High Resolution Two Dimensional Electrophoresis Of Soluble Proteins In Human Spermatozoa, Hsing-Ying Liu

Chemistry & Biochemistry Theses & Dissertations

The evaluation of human semen is very important for diagnosis of male infertility. Combined with the analysis of seminal plasma, a comprehensive biochemical screening of human spermatozoan proteins may be of assistance in providing qualitative and quantitative information to the physician to assist with evaluation.

In this study, human spermatozoa were ruptured by sonication. The soluble spermatozoan proteins were separated from the cellular debris by centrifugation and then analyzed in dissociated forms by two dimensional electrophoresis. The separated proteins were visualized by a silver stain method.

It has been shown that these combined techniques can resolve- four to five hundred …

Determination Of N,N'-Diphenylguanidine In Biological Tissues And Fluids Using High Pressure Liquid Chromatography, Edward Sidney Hunter Iii

Chemistry & Biochemistry Theses & Dissertations

N,N'-Diphenylguanidine (DPG) is a potentially toxic compound used in the vulcanization of rubber products. Distribution of DPG in hybrid mice (C57BL/6J interbred with DAB₂) was determined following intraperitoneal injection. Urine, fecal material, whole blood and tissues were collected at various times after administration of DPG and the extracts analyzed for DPG by high pressure liquid chromatography. Recovery of DPG from spiked urine and whole blood exceed 90%, however, the recovery of DPG from spiked tissue samples was approximately 70%.

DPG concentration in whole blood reached a maximum within ten minutes and rapidly decreased through 180 minutes after administration …

A Survey Of Glycosphingolipids In The Human Aorta: Their Relationship To Glycosphingolipids Of Blood And To Atherosclerosis, John L. Nappier

Dissertations

The four major glycosphingolipids, glucosyl ceramide, lactosyl ceramide, galactosyl lactosyl ceramide and globoside were isolated from both diseased and normal tissue from human aorta by solvent extraction followed by solvent partitioning and column chromatography. The levels of each were then determind quantitatively by high pressure liquid chromatography (HPLC) after derivatization. The individual glycosphingolipids were isolated by semi-preparative HPLC and the fatty acid composition of each was determined, after methanolysis, by gas chromatography.

In the diseased tissue, the amount of glycosphingolipid as well as the amount of crude lipid increased relative to the normal tissue. The relative amounts of cerebrosides increased …

Determination Of Erythrocyte Zinc Concentrations Using Zeeman Effect Atomic Absorption Spectroscopy, Tanya Maranda Lewis

Chemistry & Biochemistry Theses & Dissertations

The purpose of this research project was to develop assay procedures for determination of zinc in erythrocytes. Three methodologies were developed. The methods were evaluated on the basis of within-run and between-run precision. The most reproducible method, the 213.856 nm flame methodology, was used to determine red blood cell zinc concentrations in more than 200 samples obtained from the Neonatal Intensive care Unit at the Children's Hospital of the King's Daughters.

This study demonstrated that erythrocyte zinc levels in premature infants were similar to those of healthy adults. A linear relation between plasma and red blood cell zinc concentrations was …

Direct Optical Scanning Gel Chromatography: Improvements To The Technique And Application To Several Diverse Biochemical Problems, John Grothusen

All Dissertations

No abstract provided.

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, …

Investigation Of The Substrate Binding Site Of Nad Glycohydrolase In Human Seminal Plasma, Sau C. Cheung

Chemistry & Biochemistry Theses & Dissertations

Nicotinamide adenine dinucleotide glycohydrolase (NADase) (E.C. 3.2.2.5) from human seminal plasma (HSP) was partially purified through a multistep purification procedure, including affinity chromatography on Amicon Red Matrex Gel A. The NADase activity was assayed by a fluorometric method with nicotinamide 1,N⁶ -ethenoadenine dinucleotide as the substrate. The enzymatic cleavage of etheno-NAD by NADase was accompanied by an eight-fold increase in fluorescence. This large increase in fluorescence provided a sensitive way to follow the rate of etheno-NAD hydrolysis.

NADase was found to be less specific for the purine moiety of NAD, since etheno-NAD was accepted as a substrate. The enzyme …

Conformational Analysis Of Nonsteroidal Estrogen Analogs, Robert N. Spady

Loma Linda University Electronic Theses, Dissertations & Projects

In target tissue, estrogens initiate a series of events, ending in protein synthesis, by binding to a cytoplasmic receptor. Once the estrogen binds, the receptor complex is activated--a process including dimerization of the estrogen complex with another subunit. In order to study the possible receptor-ligand interactions, conformational analysis was performed on several nonsteroidal estrogen analogs, using the CAMSEQ8 molecular mechanics program.

The probability that a molecule will exist in a given conformational energy well depends both on the energy and the shape of the well. A method has been previously worked out to calculate probabilities using both of these variables, …

Analysis Of The Surface Polysaccharides Of R. Trifolii Su843 And Its Sym Plasmid Alterated Mutants, Jauh-Lin Duh

Masters Theses

The Rhizobium trifolii polysaccharides from SU843 and mutants--HC843(pBR1AN), HC843, 8002--in which the sym plasmid is replaced, absent, or mutated by transposon Tn5 were isolated. The surface polysaccharides of genetically altered mutants were compared quantitatively and qualitatively with one another and with the parent strain. The interpretation of these results is discussed in relation to the function of Rhizobium surface polysaccharides in the symbiotic process. The symbiotic ability of SU843 and mutant strains is as follows: (a) SU843, nod⁺, fix⁺; (b) mutant 8002, roa⁺ (still attaches to root hair), hac^-, nod^-; (c) …

The Relationship Between Whole Brain Catecholamine Depletion In Carassius Auratus And The Exposure To Inescapable Shock In A Learned Helplessness Paradigm, Roderick J. Misunis

Masters Theses

Learned helplessness is a psychological concept that describes the subsequent escape-avoidance behavior of experimental subjects who are exposed to uncontrollable stressors. Subjects after treatment are unable to respond in situations where escape is possible. Two major theorists, Seligman and Weiss, have proposed explanations concerning the phenomena. Seligman feels that the subject is unable to respond due to the fact that the exposure to an uncontrollable stressor has caused him to learn to be "helpless". Weiss feels that the inability to respond can best be described by alterations in the subject's brain neurochemistry, specifically the neurotransmitter norepinephrine. This paper is an …

Electroimmunoassay A New Competitive Protein Binding Assay Using Antibody Selective Electrodes, George Raymond Connell

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to utilize concept of antibody selective electrodes to develop a new competitive protein binding assay. This is a new concept and potentially important analytical technique because it combines the advantage of RIA (sensitivity and selectivity) with advantages of electrical assays (speed and low cost).

Electroimmunoassay: A New Competitive Protein Binding Assay Using Antibody Selective Electrodes, George Raymond Connell

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to utilize the concept of antibody selective electrodes to developed a new competitive protein binding assay. This is a new concept and a potentially important analytical technique because it combines the advantages of RIA (sensitivity and selectivity) with the advantages of electrical assays (speed and low cost). A conjugate of PGE₂ and dibenzo—18- crown-6 (a cation selective ionophore) was synthesized, and incorporated into a plastic membrane. The ionophore conjugate increased the selectivity of a polyvinyl chloride (PVC) membrane to monovalent cations. Transmembrane potential was altered by anti-PGE₂ antisera in a concentration dependent …

Development Of A Procedure For Analysis Of High Density Lipoprotein Subclasses, Chan Chin

Chemistry & Biochemistry Theses & Dissertations

Human serum high density lipoprotein subclasses, HDL₂ and HDL₃ , were isolated by preparative salt density gradient ultracentrifugation and further analyzed by electrophoresis on a 4 to 15% concentration gradient polyacrylamide gel.

The separation of the major classes of serum lipoproteins was achieved after a single ultracentrifugation for 272,000 g at 15°c in a swinging bucket rotor. High resolution concentration gradient gel electrophoresis was found to be particularly suitable for the separation of lipoproteins. The isolation of HDL by precipitation methods, heparin-manganese and dextran-magnesium have also been evaluated in this study, and were found not to completely separate …

Isomers Of 5.8s Ribosomal Rna, David Smith

Chemistry & Biochemistry Theses & Dissertations

When whole cell RNA was separated by high resolution PAG (i.e., polyacrylamide gel) electrophoresis, 5.8S rRNA migrated as three higher mobility intense bands, and three faint bands of lower mobility. The three intense bands represent conformational isomers which migrated as a single band under denaturing conditions on PAG. The three faint bands represent conformational isomers of a minor form of 5.8S rRNA which is elongated at the 5' terminus of the molecule. This minor form migrates as a single band of lower mobility than the major form when electrophoresed on a denaturing PAG. The minor form was determined to have …

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 …

Ortho Cyclization Reactions Of Catecholamines, Robert Lee Hudkins

Chemistry & Biochemistry Theses & Dissertations

A study was undertaken to examine the potential for ortho cyclization associated with the synthesis of 1,2,3,4,-tetrahydroisoquinolines using the Pictet-Spengler reaction. Specif1cally, the condensation of the biogenic amines, dopamine, norepinephrine and epinephrine were carefully examined in light of current literature and the facility and extent of ortho cyclization in each case. The para isomers of these three biogenic amine condensation products were isolated and characterized, while only the ortho isomer derived from dopamine was found stable and could be isolated, and structurally identified. These studies demonstrate that a second cyclization product does, in fact, form in each instance and that …

Nuclear Envelope Proteins: Chemical Characteristics And Molecular Interactions Of The Lamins, David Lee Cochran

Theses and Dissertations

The lamins are a group of proteins in a residual nuclear envelope fraction derived from the nuclear lamina. The absence of specific functional assays for the two predominant species, lamin A and lamin B, has complicated their biochemical characterization. Initial studies reported in this work involved the enzymic cleavage of isolated lamins A and B and another lamina protein with a molecular weight of 61,000-daltons. The results obtained with each of three enzymes indicated strong similarities between the three nuclear envelope proteins.

Further chemical characterization of the lamins has been achieved. Lamins A, B and C have been separated by …

Correlation Between Glutathione Peroxidase Activity And Selenium Concentrations In Both Plasma And Erythrocytes, Agatha Dudzai Munyanyi

Chemistry & Biochemistry Theses & Dissertations

Plasma and erythrocyte glutathione peroxidase activities and selenium concentrations were determined for 38 apparently healthy female subjects. In addition, erythrocyte lead and plasma lead and iron concentrations were determined in each subject.

Effects of age, smoking, vitamin and mineral supplementation, use of caffeinated beverages and oral contraceptives on glutathione peroxidase activity were determined and interelement relationships were investigated using the one tailed t-test and Spearman Rank Correlation Coefficients

In Vitro Enzymatic Assay Of Rna Methylation, Pamela Jean Eubanks Gallup

Chemistry & Biochemistry Theses & Dissertations

An assay procedure for in vitro enzymatic methylation of mannnalian ribosomal RNA has been developed in this study. The assay procedure, utilized for the comparison of normal and neoplastic methylase activities (using mouse liver and Ehrlich ascites cells as sources of enzyme), is a modification of previously published methods (52,53). A 100,000 x g supernatant (SlOO) enzyme preparation was incubated with 28S-5.8S rRNA and tritium-labeled S-adenosyl-L-methionine. The RNA was extracted, applied to DEAE cellulose paper, washed, and the radioactivity counted. The neoplastic cell methylase preparation was more active in methylating both exogenous neoplastic and normal 28S-5.8S rRNA than the normal …