Hybridoma cells utilize glutamine as their primary nitrogen source and excrete ammonia as a metabolic waste product. This ammonia can quickly accumulate to toxic levels in hybridoma culture media, and can severely reduce monoclonal antibody production (Ozturk et al., 1991). The enzyme glutamine synthetase (E.C. 6.3.1.2), which catalyzes the reaction UNFORMATTED EQUATION FOLLOWS: NH₄⁺ + L-glutamate + ATP Mg²⁺ → L-glutamine + ADP +Pᵢ +H⁺, UNFORMATTED EQUATION ENDS was evaluated as a means of reducing ammonia and replenishing glutamine in hybridoma culture medium. The effect of each enzyme reactant on soluble glutamine synthetase activity was quantified, and enzymatic reaction equilibrium evaluated. Enzyme reaction rates in two culture media, both with and without serum, were compared. Glutamine synthetase was immobilized via three different methods, and their effects compared. Cell sensitivity to each enzymatic reactant was studied. Finally, immobilized glutamine synthetase was incorporated in a hybridoma cultivation, and its effect on culture characteristics evaluated.

The RecA protein of Escherichia coli is a small protein involved in many important functions including homologous recombination, mutagenic repair, regulation of the SOS system, and prophage induction. In normally growing cells, RecA is in an inactive form. However, when cells are subjected to DNA damage, RecA forms a helical filament with single stranded DNA and ATP. This ternary complex is the activated form of RecA. A key step in the regulation of several of these processes is the RecA mediated cleavage of different proteins. However, RecA is not a classic protease, but instead causes these proteins to undergo autodigestion. The main goal of this research was to investigate the role that RecA plays in cleavage by determining what residues in RecA interact with the cleavage substrates LexA, UmuD and λ CI. A possible model for the binding of the cleavage substrates in the cleft formed between two adjacent RecA monomers in an activated filament has been proposed. Site-directed mutagenesis was used to change residues in RecA that map to the cleft. An analysis of previously characterized recA mutants also suggested other regions of RecA that might interact with the cleavage substrates. Site-directed mutagenesis was used to also change these residues. Candidate residues were changed to alanine in order to reduce side chain contacts while minimizing perturbation of protein folding. The RecA mutant proteins were then characterized for ability to do recombination and DNA repair, and were examined for ability to mediate the cleavage of LexA, UmuD and λ CI. Several mutants showed some defects in cleavage of LexA or λ CI, while being proficient for other RecA functions. The fact that these mutants are selectively defective for cleavage of one protein, but not the others, suggests they can form activated filaments. Most of the mutations that differentially affected cleavage of LexA or λ CI mapped outside of the cleft region. This result suggests that the cleavage substrates do not bind in the cleft, but instead that the cleavable proteins may bind in the groove formed between turns of the RecA filament.

<p> While ribonuclease A is a well studied enzyme, the catalytic role of an active site lysine (Lys41) is not understood, as Lys41 was suggested to interact with the scissile phosphate of the substrate and/or interact with the 2&rsquo;-hydroxyl nucleophile. To distinguish these models, we sought to use a double mutant cycle analysis. Lys41 was mutated to alanine and arginine, and the phosphate oxygens were individually substituted with sulfur. Challenges were encountered including obtaining sufficient quantities of pure enzyme and reproducible kinetics results. Nevertheless, our results show the Sp phosphorothioate decreased activity &sim;10-fold, while the Rp substitution led to a &sim;300-fold decrease. Structures show the pro-Rp oxygen situated to interact with Lys41, and an important role for this interaction in catalysis is supported by the larger deleterious effect from substitution at the Rp site. The results provide a foundation for using double mutant cycle analysis to investigate ribonuclease A catalysis.</p>

<p> Alzheimer&rsquo;s disease (AD) is the most common cause of dementia among older people characterized by low concentration of acetylcholine in the brain. Butyrylcholinesterase (BChE) is one of the two cholinesterase enzymes that catalyze the hydrolysis of acetylcholine into choline and acetic acid, and its activity is found to be increased above normal in AD patients. Therefore, BChE inhibitors may help to increase acetylcholine concentration in the brain and hold promise in the treatment of the disease. To search for the most effective BChE inhibitors, we were interested in the synthesis of structurally different organophosphates and their interaction with BChE.</p><p> A variety of organophosphates divided into three main types of scaffolds were synthesized and evaluated in vitro for their activity against BChE. Di-n-butyl aryl phosphates (DBAPs) were found to be selective inhibitors of BChE, especially when placing methyl groups on the phenyl ring. Racemic and optically active alkyl aryl cholinyl phosphates (AACPs) were successfully synthesized by using the methods we developed in our laboratory, and these analogs were stronger BChE inhibitors than DBAPs, particularly when the alkyl chain was lengthened. Also, a library of tetraalkyl bisphosphates with both oxygen and sulfur containing linkers were synthesized and their inhibitory abilities examined. At this time, we observed that the six carbon chain is the optimal linker chain length and the sulfur atom containing linker exhibited higher inhibition than the oxygen atom containing linker.</p>

Results of characterization of short DNA using nuclear magnetic resonance are described. Nuclear magnetic resonance studies indicate that double stranded DNA is not a static structure. Many types of internal motions are present within the DNA chain, some being strongly coupled, while others are uncoupled or coupled. The coupled motions probably produce wave-like motions observed at low DNA concentrations. The uncoupled motions are more localized, reflecting the pseudorotational qualities of the deoxyribose ring. The sugar carbons C2' and C5' behave in a unique manner, with some motions of C2' being completely uncoupled from other internal DNA motions, while the motions of C5' are highly coupled. These results suggest a possible concentration dependent change in configuration of the sugar ring from C2' endo to C3' endo, where some of the internal motions of the sugar ring are uncoupled from motions of the DNA backbone. A model of conic diffusion superimposed on axially symetric rotation suggests that the rotational correlation times for the internal motions of double stranded DNA are approximately four nanseconds. Solutions of short DNA chains become ordered at high concentrations in a highly cooperative manner, with the critical concentration being inversely related to the chain length. NMR resonance intensities are greatly reduced above the phase transition. NMR resonances are also repressed for long DNA when compared with those of 147 np at the similar concentrations below the phase transition. These results suggest that any inability to observe ('13)C NMR resonances from double stranded DNA is due to local interchain interactions which "freeze out" the coupled internal motions. The internal motions of the carbons are greatly restricted when the DNA is incorporated into nucleosome core particles. However, the motions of the DNA phosphates are not greatly / affected. The ability to obtain ('13)C NMR spectra from short double stranded DNA allows for a wide range of new experiments such as studies of DNA-environmental interactions. / Source: Dissertation Abstracts International, Volume: 43-04, Section: B, page: 1076. / Thesis (Ph.D.)--The Florida State University, 1982.

Thermolysin is a thermostable neutral zinc endopeptidase with a molecular mass of 34.6 kDa. The stopped-flow fluorescence technique has been used to investigate the catalytic pathway for the hydrolysis of dansyl-peptides by thermolysin. The origin of the fluorescence changes observed during the hydrolysis of dansyl-substrates by thermolysin has been investigated. Depending on the substrate, these changes arise from changes in the concentration of the substrate (F$\sp{\rm SP}$) or the changes in the concentration of ES$\sb{\rm i}$ as reflected by resonance energy transfer (F$\sp{\rm RET}$). Both F$\sp{\rm SP}$ and F$\sp{\rm RET}$ changes have been used to monitor the reaction process, but different mathematical formalisms have been used to determine the kinetic parameters and the results from all are in good agreement. The F$\sp{\rm RET}$ contribution correlates strongly with the K$\sb{\rm M}\sp{-1}$ values for these reactions, as expected for a process that monitors the (ES$\sb{\rm i}$). The effect of sodium halide salts on the optical and emission properties of thermolysin and its dansyl substrates has been studied. The effect of these salts on the activity of thermolysin is substrate dependent. The main effect is to reduce the K$\sb{\rm M},$ indicating that chloride increases the strength of substrate binding. For some substrates, k$\sb{\rm cat}$ is compensatorily reduced, while for others it is unchanged. The effects of these salts can be described by a general modifier mechanism. Low-temperature stopped-flow fluorescence measurements have been carried out to study elementary steps that occur during the pre-steady-state time region reaction of thermolysin with Dns-Gly-Phe-Ala. A new relaxation characterized by an increase in dansyl fluorescence and decrease in Trp fluorescence has been observed. This corresponds either to an isomerization of thermolysin or to the interconversion of two ES$\sb{\rm i}.$ / Source: Dissertation Abstracts International, Volume: 53-11, Section: B, page: 5706. / Major Professor: Harold E. Van Wart. / Thesis (Ph.D.)--The Florida State University, 1992.

Metabolism of the extracellular matrix (ECM) is important in many normal physiological processes, such as wound healing and aging, as well as in many disease states, such as rheumatoid arthritis and cancer cell metastasis. Collagen is the major protein component of the ECM, and comprises one-third of the total protein found in mammalian organisms. Two distinct collagenases are known to exist in humans, human fibroblast collagenase (HFC) and human neutrophil collagenase (HNC). These collagenases are members of a family of ECM degrading enzymes called the matrix metalloproteinases (MMP). The MMP are secreted from cells as inactive zymogens which, subsequently, become activated in the extracellular milieu. / The zinc content of five human pro-MMP has been quantitated, and the role of zinc in these MMP has been assessed. Each of these MMP is found to contain one zinc atom which is catalytically important. The catalytic zinc atom of HFC is shown to be sequestered in the latent state and exposed in the active state. Studies of the activation and inhibition of HFC and HNC by seven classes of agents reveal a common biochemical theme and multiple active forms. Two pathways are identified in the activation of HFC and HNC, chemical modification of a cysteine sulfhydryl and removal of the N-terminal propeptide. The key sulfhydryl group is found to be sequestered in the latent state of pro-HFC and is released from this sequestered environment upon activation. A model is proposed, the "cysteine switch" model, which accounts for the multiple modes of HFC activation and for the observed properties of the latent and active forms. The implication of the cysteine switch mechanism of activation for the MMP family in vivo is that it permits flexibility and selectivity in the control of the enzymes responsible for metabolism of the ECM. / Source: Dissertation Abstracts International, Volume: 53-11, Section: B, page: 5704. / Major Professor: Harold E. Van Wart. / Thesis (Ph.D.)--The Florida State University, 1992.

Six distinct collagenases present in the culture filtrate of Clostridium histolyticum have been purified to homogeneity and characterized in detail. To accomplish this end, an accurate and convenient assay for collagenase activity based on the hydrolysis of soluble {('14)CH(,3)}collagen has been developed. This assay, which can detect nanogram quantities of collagenase, has greatly facilitated the purification. The key step in the isolation of the individual collagenases, which have very similar physicochemical properties, is chromatography over a red dye-ligand column. The purified enzymes are homogeneous by sodium dodecyl sulfate gel electrophoresis and are devoid of any contaminating enzymatic activities. / The six collagenases, designated (alpha), (beta), (gamma), (delta), (epsilon), and (zeta), have been characterized as to their molecular weights, isoelectric points, carbohydrate content and amino acid compositions. Atomic absorption spectroscopy shows that all of the enzymes contain one gram atom of zinc per mole of protein, and variable amounts of calcium. The activities of the enzymes towards collagen, gelatin, and several synthetic peptides have been determined. These activities, taken together with other data, strongly support the assignment of the collagenases into two distinct classes. / The relationship between the individual collagenases has also been examined. Chemical modification reactions establish that all six enzymes contain essential lysine, tyrosine, and carboxyl residues. Moreover, all six enzymes cross react with antiserum prepared against (beta)-collagenase. Information about the sequence relationships between the enzymes has been obtained by comparison of their tryptic digestion and of their cyanogen bromide reaction products. The results of these experiments indicate that the collagenases within each class have extensive sequence homology with each other, but that there is little sequence homology between the two classes. In addition, the data show that (beta)-collagenase probably consists of domains that have homologous amino acid sequences. Gene duplication is implicated as a possible evolutionary mechanism for the development both of two classes of enzymes and of homologous domains in (beta)-collagenase. / Source: Dissertation Abstracts International, Volume: 44-11, Section: B, page: 3384. / Thesis (Ph.D.)--The Florida State University, 1983.

The core histone (H2a, H2b and H3) mRNAs, whose levels are correlated with DNA synthesis, were studied in mouse cells by S1 nuclease analysis. These mRNAs are transcribed from a family (10-20 members) of genes that have highly conserved protein coding regions but vary extensively in the transcribed, untranslated regions. Thus, the products of individual genes can be readily distinguished by the S1 nuclease assay. Eight histone genes from 3 mouse histone gene clusters (MM221, MM291 and MM614) were used as probes. One of these clusters--MM614--encodes about 40% of the H2a or H3 mRNA found in cultured mouse cells, while the other 2 clusters--MM221 and MM291--encode only 5-10% of the H2a, H2b or H3 mRNA in the same cells. MM614 maps to chromosome 3, while MM221 and MM291 map to chromosome 13. Although the relative contribution of a specific gene to the family of histone mRNAs is the same in several different cultured mouse cell lines and 18-day fetal mouse cells, a different situation is observed in mouse eggs and blastocysts. In blastocysts, the H3 mRNAs encoded by the three clusters are of similar relative abundance--about 5%, while in eggs the H3-614 and the H3.2-221 mRNA represent 40% and 25% of the H3 mRNA, respectively. / The levels of all of these histone mRNAs are reduced about ten fold, when mouse cells are treated with DNA synthesis inhibitors. This decay occurs with a half-life of twelve minutes. The transcription of this family of genes is altered within ten minutes after experimental inhibition or initiation of DNA synthesis. Both of these effects can be readily reversed either by reinitiation of DNA synthesis or by inhibition of protein synthesis. The relative abundance of specific histone mRNAs to the family of histone mRNAs detected does not change. This demonstrates that the MM614 histone mRNAs are transcribed at higher rates than are the MM221 or MM291 mRNAs. The data are not consistent with a tight coupling between DNA synthesis and histone mRNA metabolism but suggest that the correlation of the two processes may be mediated by deoxynucleotide metabolism. / Source: Dissertation Abstracts International, Volume: 45-04, Section: B, page: 1176. / Thesis (Ph.D.)--The Florida State University, 1984.

Rat ceruloplasmin (rCp) has been labeled with rhodamine B isothiocyanate (RBITC). RBITC-labeled rCp (RBITC-rCp) was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy. Film negatives of blood smears were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound on the plasma membrane. Binding of RBITC-rCp to erythrocytes and platelets was saturable, reversible, and specific as only rCp could displace it. In both cases trypsin-treated cells showed greatly reduced binding. Applying the same technique to rat lung tissue, bronchial muscle demonstrated evidence of a receptor. / An electrophoresis mobility shift assay (EMSA) was developed to study the binding of rCp to its erythrocyte receptor. $\sp{125}$I-rCp, native polyacrylamide gel electrophoresis (PAGE), and detergent-solubilized plasma membrane were required. Triton X-100 extracted receptor bound apo-rCp as well as native rCp and had a M$\sb{\rm r}$ of 150,000. The binding of $\sp{125}$I-rCp was specific as only rCp could displace it. The receptor extracted with SDS detergent showed activity after 80$\sp\circ$C for 10 min, so SDS-PAGE was used to separate the membrane proteins. Three classes of binding proteins were detected: (1) nonspecific irreversible, (2) nonspecific reversible, and (3) specific and reversible--the receptor. Using this technique the M$\sb{\rm r}$ for the receptor was less than 80,000. / The reason chloroethylamine-treated agarose binds Cp from plasma preferentially has been elucidated. Polyethylenimine tentacles are formed which require a uniform negative surface charge for proteins to bind. / Source: Dissertation Abstracts International, Volume: 53-07, Section: B, page: 3461. / Major Professor: Earl Frieden. / Thesis (Ph.D.)--The Florida State University, 1992.