Enzymes and Coenzymes Commons^™

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 …

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 …