Enzymes and Coenzymes Commons^™

Cellular Responses In Escherichia Coli To Lethal And Sublethal Doses Of Ozone, Indira Ruth Komanapalli

Loma Linda University Electronic Theses, Dissertations & Projects

Ozone is a major component of photochemical smog. High levels of this pollutant, sufficient to affect human health are found in many urban areas worldwide. Though limited studies in humans are supported by extensive findings from animal experiments, a difficulty in interpreting the results of these experiments has lead to an ambiguity on the biochemical mechanism of ozone toxicity. To elucidate the mechanism by which ozone causes cell damage and eventual cell death we conducted a comprehensive study using Escherichia coli K-12 as a model.

Studies on the comparative inactivation of bacteriophage lambda (λ), Escherichia coli, and Candida albicans …

An Enzyme-Linked Immunosorbent Assay For The Detection Of Cardiac Myoglobin Using Monoclonal Antibodies, Deborah F. Kelly

Chemistry & Biochemistry Theses & Dissertations

The most prevalent life-threatening disease worldwide, secondary to coronary artery thrombosis is Acute Myocardial Infarction (AMI). Increases in three clinical markers have been explored as an early means of AMI detection: Cardiac myoglobin, and the two specific cardiac isoforms of Creatine Kinase, CK-MM and CK-MB. However, myoglobin levels have shown to give a more accurate correlation (R = 0.89) than does Creatine Kinase evaluation. Therefore, we have developed a two-site Enzyme-Linked Immunosorbent Assay (ELISA) using monoclonal antibodies against myoglobin isolated from human heart tissue. This assay is based on specific anti-genantibody interactions and is sensitive at the nanogram level. We …

Competitive Enzyme Immunoassay Of Catechin In Human Blood Serum, Jay Fedorowicz

Chemistry & Biochemistry Theses & Dissertations

( +)-Catechin is a type of polyphenolic compound, and it is enriched in fruits, plants, and various beverages. Polyphenolic compounds also play an important role in the human diet. Many scientists, especially wine researchers, have proposed that(+)catechin and its polymers could have many positive health effects. Thus, it is essential to learn more about (+)-catechin and other polyphenolics. Presently, high performance liquid chromatography is the only method to detect ( + )-catechin levels in human blood serum, but this method has the high cost of instrumentation and does not provide good sensitivity. The objective of this project is to develop …

Ornithine Decarboxylase And Polyamines In Perinatal Hypoxia, Satyaseelan Packianathan

Loma Linda University Electronic Theses, Dissertations & Projects

Hypoxic and hypoxic/ischemic episodes contribute to various neurological aberrations, learning disabilities, et cetera in surviving infants. Despite major advances in understanding the pathophysiology of hypoxia, the cellular basis for hypoxic-mediated injury remains poorly understood. In addition, the role of developmentally essential enzymes vis á vis their contributions, or lack thereof, to hypoxic-mediated injury has not been adequately investigated. In the series of studies comprising this dissertation, we tested the hypothesis that acute and/or chronic hypoxia affect the activity of omithine decarboxylase (ODC; E.C. 4.1.1.17), the developmentally essential enzyme in polyamine biosynthesis, in the fetal and newborn rat brain. Fetal and …

A Physical Map Of The Klebsiella Pneumoniae M5a1 Genome, Alexandrine Randriamahefa

Loma Linda University Electronic Theses, Dissertations & Projects

Klebsiella pneumoniae is an enteric opportunistic pathogen which commonly causes pulmonary, urinary tract and kidney infections. It has also been widely used for the study of nitrogen fixation. In this study, a macrorestriction map of K. pneumoniae M5a1 chromosomal DNA was constructed using pulsed field gel electrophoresis (PFGE) and Southern hybridization techniques. The genome DNA was digested with two rare cutting restriction enzymes (XbaI and BlnI) and the genome size was estimated from the sum of the fragment sizes as 5,121 Kb (+2.8%). A tentative order of fragments was deduced using Southern hybridization with purified restriction fragments. …

Longitudinal Study Of Plasma And Erythrocyte Selenium Levels In Type I Diabetic Children During Development And Treatment Of Ketoacidosis, Pi-Hsia Fan

Chemistry & Biochemistry Theses & Dissertations

Most studies on the biological activity of selenium have focused on its role at the active site in the ubiquitous enzyme, glutathione peroxidase (GSH-Px) which catalyzes the removal of hydrogen peroxide and organic hydroperoxides (1). A connection between selenium deficiency and a wide range of diseases in animals and humans has been reviewed (2). Development of various forms of human cancer (3), failure of the immune system (4), as well as some cardiovascular diseases (5) have been reported or suspected to correlate with selenium deficiency. However, the physiological function of selenium in these different diseases is not fully understood, and …

Kinetic Characterization Of A Recombinant C-Terminal Mutant Of Reverse Transcriptase From The Human Immunodeficiency Virus, Thomas S. Heard

Chemistry & Biochemistry Theses & Dissertations

The human immunodeficiency virus (HIV) reverse transcriptase (RT) (EC 2.7.7.49) is the central replication enzyme for HIV. In general, the kinetic mechanism for this and all other polymerases involves the ordered binding of two substrates: a primer-template (PT) followed by a deoxyribonucleoside triphosphate (dNTP). Previous investigations prompted this research when it was discovered that the substrate dNTP, in absence of PT, could protect a recombinant c-terminal mutant HIV-1 RT from inhibition by pyridoxal-5'-monophosphate (PLP), an active-site dNTP inhibitor. In contrast, the non-mutant recombinant HIV-1 RT required both substrates for protection from PLP inhibition. This investigation sought to determine if this …

The Expression And Regulation Of The Hsdk Genes Of Escherichia Coli K-12, Ainu Parkash

Loma Linda University Electronic Theses, Dissertations & Projects

The DNA restriction and modification (R-M) genes in bacteria play a defensive role in the organism, offering protection to the host from invaders such as bacteriophages. The expression of these genes in E. coli K-12 had not previously been studied due to the complexity of the EcoK R-M enzymes. The use of lacZ gene fusion techniques allowed studies of the expression of the hsdK genes for the first time. Operon fusions of Mu(/ac) to the hsdK genes show that the two hsdK promoters, p _res and p _mod, are weak (50 Miller units) and that …

The Effects Of Glutathione On Human Keratinocyte, Manisha N. Trivedi

Theses and Dissertations in Biomedical Sciences

Cultured human keratinocytes were grown in vitro to assess the effects of glutathione, a thiol, on human keratinocyte proliferation. Single keratinocytes were harvested from newborn (greater than 5 days old) foreskin and were grown in culture flasks such that experiments could be performed in approximately 2 to 3 weeks. In order to evaluate the effects of glutathione (GSH) on keratinocyte growth, experiments were performed to determine the growth curve, plating efficiency, and incorporation of radiolabelled thymidine. Growth studies, performed with different concentrations of glutathione, revealed that 0.065 μM was the most effective concentration when compared to control cell cultures, and …

Activation In Vitro Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Panayota Eriotou

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to isolate and purify the enzymes, transfer RNA-guanine ribosyltrasferase and protein kinase C, and to determine whether the phosphorylating enzyme activates the insertion enzyme in vitro. Transfer RNA-guanine ribosyltransferase lost activity within several days after isolation and total, complete reactivation was accomplished in the presence of protein kinase C. This demonstrated that ribosyltransferase's instability is related to the degree of its phosphorylation. It is proposed that modification of tRNA is controlled by protein kinase C. Deactivation of the insertion enzyme leads to hypomodified tRNA which in turn is associated with neoplasia. Potential use …

Microwell Based Solid Phase Competitive Enzyme Immunoassay For Estradiol And Estriol, Jeng-Ting Yang

Chemistry & Biochemistry Theses & Dissertations

A sensitive and simple microwell based competition enzyme immunoassay for the quantitative determination of estradiol (E₂) and estriol (E₃) was developed. Nicrowells coated with antibody were incubated with antigen followed by adding horseradish peroxidase (HRPO) conjugate. The assay, which can be performed within two hours at room temperature, involved simultaneous incubation of E₂ - or E₃-HRPO conjugate and serum sample in polystyrene microwells coated with anti- E₂ or anti-E₃ gamma globulin fraction.

Gamma globulin was isolated from whole anti-serum by DEAE-cellulose chromatography. A carbodiimide coupling method was utilized to prepare the …

Development Of A Solid Phase Enzyme Immunoassay For Total Serum Testosterone, Li Li

Chemistry & Biochemistry Theses & Dissertations

An enzyme-labeled immunoassay of sufficient range and sensitivity was developed for serum testosterone determination. In this method, the testosterone in the serum sample and testosterone-HRPO conjugate compete with each other for the limited antibody binding sites on the anti-testosterone gamma globulin coated microwell. The assay can be performed in only 2.5 hours.

The gamma globulin fraction was isolated from the anti-testosterone whole serum by DEAE-cellulose column chromatography. Testosterone-peroxidase conjugate was prepared by a carbodiimide coupling method. The interference which arises from the binding of testosterone to steroid hormone binding protein was eliminated by the addition of 17 beta-estradiol. All assay …

Regulation Of Queuine Insertion Into Transfer Rna: Effects Of Tumor Promoters, Bonnie Jean Brooks

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to treat normal human fibroblasts with known tumor promoters and observe queuine modification of tRNA. The queuine insertion enzyme, tRNAguanine ribosyltransferase, was studied in vivo and in vitro. Tumor promoter-treated human fibroblast cultures exhibited variable queuine-insertion rates with a transient inhibition that correlated with variable levels of queuine modified tRNA over time in culture from passages 3 — 8. In contrast the in vitro studies showed that phorbol cetera and saccharin actually increased insertion with strong evidence indicating phosphorylation as a positive modulating force of the enzyme activity. It is proposed that chronic stimulation …

Effect Of Photoperiod On Developmental Morphology And Enolase Isoenzyme Immunohistochemistry In Rat And Djungarian Hamster Superficial Pineal Glands, Chalmer D. Mcclure

Loma Linda University Electronic Theses, Dissertations & Projects

The best understood functional activity of the pineal gland is its diurnal production of melatonin in response to environmental lighting cues. Several enzymes of the melatonin pathway respond to daily photoperiod changes, for example hydroxyindole-O-methyltransferase (HIOMT) and serotonin N-acetyltransferase (SNAT). Increased levels of the glycolytic enzyme neuron-specific enolase (NSE) are thought to reflect increased physiological demands placed on neurons and neuroendocrine tissues. Homodimer non-neuronal enolase isoenzyme (NNE) is immunolocalized to cells, and the hybrid enolase (consisting of subunits from NSE and NNE) has been seen in cerebellar stellate and basket cells. Although not rate limiting, concentrations of both NSE and …

Hypoxanthine-Induced Differentiation Of Cultured Human Leukemia Cells, Gayle Jennette Singleton

Chemistry & Biochemistry Theses & Dissertations

Human cultured leukemia cells appear to have a decreased amount of inosine in their tRNA. When cells with inosine deficient tRNA are placed in a hypoxanthine fortified media, they incorporate hypoxanthine into their tRNA by the action of the enzyme tRNA-hypoxanthine ribosyl transferase. This generates the nucleoside inosine in the tRNA. The cultured human leukemia cell lines, CCRF-CEM, HL-60, and HGPRT(-) HL- 60, incorporate hypoxanthine into their tRNA, as determined by tRNA isolation, hydrolysis, and HPLC analysis. Hypoxanthine treatment dramatically inhibited cell growth in conjunction with partial induction of differentiation in the CCRF-CEM, HL-60, and HGPRT ( - ) HL-60 …

An Analysis Of Protein-Ligand Interactions For Enzyme Ii-Mannitol From Escherichia Coli, Brent Lee Wood

Loma Linda University Electronic Theses, Dissertations & Projects

Enzyme II^mtl of Escherichia coli was purified from mtlA-overexpressing E. coli strain LJ1112 extraction of isolated membranes with deoxycholate, followed by hexyl agarose and ω-amino hexyl agarose chromatography in Lubrol PX. A 10-fold increase in total pure protein and 2.5-fold increase in specific activity over previously reported procedures was obtained. Tryptic fragments of Enzyme II^mtl were also purified and identified as the membrane and cytoplasmic domains by protein sequencing and SDS-PAGE. The fluorescence spectrum of Enzyme II^mtl suggested that two populations of tryptophan residues existed with emission maxima at 322 and 344 nm, corresponding to buried …

Synthesis And Characterization Of N-Alkyl-Adenosine-5'-Diphosphates, Han-Ping Huang

Chemistry & Biochemistry Theses & Dissertations

n-Alkylphosphates and adenosine derivatives have been observed to be competitive inhibitors of coenzyme molecules in many NAD⁺ requiring enzymes. In earlier studies, adenosine derivatives such as AMP, ADP and ADP-ribose were found to be coenzyme competitive inhibitors of the Lactate Dehydrogenase catalyzed reaction. However, n-alkylphosphates, to the contrary, + were found to be noncompetitive inhibitors of NAD⁺.

Furthermore, it was observed previously in Dr. Yuan's laboratory that the effect of n-octylphosphate on the LDH catalyzed reaction was changed from noncompetitive to competitive in the presence of low concentration of AMP. Rossmann et al. reported that the binding …

Determination Of Human Sperm Cell Acrosin Activity From Normal And Subfertile Men, Dilrowshan H. Haque

Chemistry & Biochemistry Theses & Dissertations

Acrosin is an acrosomal enzyme which is reported to be involved in sperm penetration through cumulus, zona, and ooplasma. This enzyme has been reported to be significantly lower in sperm of infertile men compared to sperm of fertile men. The acrosin assay which has been developed can be used for studies in patients with suspected infertility and may be used on a fraction of most semen specimens being evaluated in a clinical andrology laboratory.

Within-run precision studies yielded coefficients of variation of 11% for a low-activity pool and 9% for normalactivity pool specimens. Between-run precision studies using a single donor …

An Immunochemical Analysis Of Alcoholic Hyaline And Its Relationship To Keratin Intermediate Filaments, David L. Drexler

Loma Linda University Electronic Theses, Dissertations & Projects

Alcoholic Hyaline (AH) and keratin intermediate filaments possess ultrastructural, biochemical and immunochemical similarities. This study compares human AH, human stratum corneum keratin (HSCK) and normal human liver cytokeratin (HCK) by quantitative immunochemical means, using polyclonal as well as monoclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) were employed, as well as "immunoblotting" and immunocytochemical techniques. HCK was isolated using Triton X-100 and "high-salt” buffers and exhibited seven major polypeptide bands on SDS-PAGE (41-58 kd). Antisera were made toward HCK and the two major HCK bands. Other immunochemicals included antisera toward AH, the three major AH bands, and HSCK, as well as 3 …

Partial Purification And Characterization Of F₄₂₀-Dependent Nadp Reductase From Methanobrevibacter Smithii Strain De1, Scott D. Sheridan

Dissertations and Theses

The F₄₂₀-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum …

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 …

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 …

Development Of An On-Line Computer System For Enzyme Kinetics Studies, Jacqueline R. Thweatt

Chemistry & Biochemistry Theses & Dissertations

The PDP-11/10 minicomputer was interfaced to the Beckman 25 ultraviolet/visible spectrophotometer and this on-line computer system was applied to enzyme kinetics studies. This interface involved hardware equipment to locate, amplify, and transmit the analog signal to an analog-to-digital converter, and software to acquire and store the digital data. Data processing programs were used to calculate kinetic parameters and statistical information from the acquired data.

The Varian Cary 219 ultraviolet/visible spectrophotometer can be readily interfaced to the Apple II Plus microcomputer by hardware equipment which can be purchased from the manufacturer. Various software may also be purchased for use in enzyme …

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 …

The Synthesis And Biochemical Evaluation Of Potential Acetylcholinesterase Reactivators, Andrea Mcdearmid Lunsford

Chemistry & Biochemistry Theses & Dissertations

Objectives of this research were to (1) synthesize, and (2) evaluate new compounds which would be potential reactivators of organophosphate-inhibited acetylcholinesterase.

A series of 1,8-diazafluorenone and 2,2'bipyridylketone oximes and oxime methiodides were synthesized (11, 13, 14, 22, 24). These compounds were developed as structural analogs of 2-PAM (2-Pyridinealdoxime methiodide) and TMB-4 (1,1Trimethylene-Bis(4-formylpyridinium bromide)dioxime), the current drugs of choice for treatment of organophosphorous poisoning.

Acetylcholinesterase from electric eel was inhibited with DFP (diisopropylfluorophosphate), an irreversible-type inhibitor, and the inhibited enzyme was treated with the various reactivator compounds. The reactivation assays were monitored with pH stat apparatus, and the data were evaluated …

Multiple Forms Of Nicotinamide Adenine Dinucleotide Glycohydrolase From Bull Semen, Stanley D. Yeatts Ii

Chemistry & Biochemistry Theses & Dissertations

Nicotinamide adenine dinucleotide glycohydrolase (NADase) of bull semen is an extracellular, soluble enzyme capable of catalyzing the hydrolysis of the nicotinamide N-ribosidic linkage of NAD⁺. In search for a rapid, simple procedure for the preparation of this enzyme, it was found that Matrex Gel Red A (Amicon) column packing served very well as an affinity gel for NADase. The enzyme was applied to the Matrex Red A column (21 cm x 1.5 cm) in 10 mM sodium phosphate buffer, pH 6.0. Surprisingly, two fractions of NADase activity were obtained when the column was eluted with a sodium chloride …

Human Placental Alkaline Phosphatase And Steroid Metabolism By Rat Adrenals, Allen G. Meier

Loma Linda University Electronic Theses, Dissertations & Projects

Human placental alkaline phosphatase was purified by butanol extraction, methanol precipitation and chromatography on DEAE-cellulose. The pH optimum was found to be 10.9. The enzyme was competitively inhibited by di-ethyl-p-nitrobenzyl-phosphonate and copper sulfate. An increase in enzymatic activity occurred in the presence of the phosphate ion (HPO₄) and ATP.

The enzyme was crystallized from a 40% saturated solution of ammonium sulfate and shown to be homogeneous by sedimentation analysis. Homogeneity of the enzyme was also demonstrated using poly-acrylamide-gel-electrophoresis. At a pH of 7.5% the electrophoretic pattern yielded a single band and showed no significant contamination.

The metabolic transformation …

Kinetic And Binding Studies On L-∝-Glycerophosphate Dehydrogenase, Paul Richard Rosevear

Chemistry & Biochemistry Theses & Dissertations

The properties of the coenzyme, NAD, binding site of chicken muscle L-∝-glycerophosphate dehydrogenase were studied. Adenosine monophosphate, adenosine diphosphate and adenosine diphosphoribose were sha.vn to inhibit the enzyme competitively with respect to NAD. The presence of adenosine, pyrophosphate and ribose regions in the coenzyme binding site of the enzyme was suggested by the inhibitor constants obtained for these compounds.

Disodium monoalkyl phosphates, n-butyl to n-dodecyl phosphate, inclusive, were also shown to inhibit the L-∝-glycerophosphate dehydrogenase catalized reaction competitively with respect to NAD. A positive chain length effect was observed in the binding of these compounds to the enzyme, suggesting the …

The Transfer Of Hydrogen In The Reduction Of Progesterone, William H. Kersey

Loma Linda University Electronic Theses, Dissertations & Projects

Experimental female rats were given daily injections of synthetic estrogens for six months. The effect on rat ovarian 20α-OH-SDH was not definitive. Diethylstilbestrol and 17α-ethynylestradiol caused a significant drop in activity as compared with controls while mestranol caused a slight increase in activity. A synthetic progestin, medroxyprogesterone, had no apparent effect on the activity of the enzyme.

Rat ovarian 20α-OH-SDH was purified fivefold by ammonium sulfate fractionation and chromatography on DEAE-cellulose.

The homogeneity of the enzyme was checked by means of disc gel electrophoresis. At pH 8.2 no significant contamination appeared in the electrophoretic pattern, but at pH 9.0 five …

The Future Of Enzyme Research, Linus Pauling

Henry Ford Hospital Medical Journal

No abstract provided.