Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for a particular gene at a single locus. The number of alleles and the extent of their variation is of particular importance due to the potential for the exploitation of genetic variation through breeding. Also, allelic variation has implications for the manipulation of gene function via genetic engineering. Pyrophosphate dependent phosphofructokinase (PFP) is considered a key regulatory enzyme involved in sucrose biosynthesis, which may provide a target for genetic manipulation to increase sucrose yields in sugarcane. The enzyme is composed of a regulatory (alpha) and catalytic (beta) protein subunit encoded by the PFP-a and PFP-p genes respectively. The PFP-p gene, which has been shown to be a single locus gene in other plant species, was used in this study as a model for allelic variation in sugarcane. Two main areas of investigation involved genomic and expression analyses to further characterise the gene. Polymerase Chain Reaction (PCR) using specific primers previously designed from conserved regions of the PFP-p gene from different plant sources, were used to amplify across exons 10 to 12 of the sugarcane PFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bp respectively, were obtained from commercial cultivars N19 and N21. Numerous clones of the fragments were obtained and sequenced. International database searches confirmed that both amplicons were identifiable as PFP-p. Comparative sequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bp fragments were poorly homologous to each other, with higher regions of homology residing in the putative exon regions (77-78%) compared to the intron regions (34- 56%). Although minor sequence variation was detected within the amplicon populations, it was evident that two major variants of the PFP-p gene are present in sugarcane. Southern hybridisation analysis revealed a simple banding pattern for PFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding to the PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicates that both variants are also present in the ancestral sugarcane germplasm and maintain the same level of heterozygosity. The presence of both gene forms in the ancestral and commercial germplasm prompts speculation that the two variants may not segregate. This theory, together with the simple Southern hybridisation pattern obtained for PFP-p, leads to the hypothesis that the two gene forms are at separate loci in the sugarcane genome, which may be closely linked on the same chromosome. The expression of the variants was investigated during different stages of sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)- peR approach. A single, identical transcription product was isolated from these and other selected tissues of the plant. In addition, the same transcript was obtained from the ancestral species representatives of modern sugarcane, Saccharum officinarum and Saccharum spontaneum. Sequence comparison of the transcribed product and the derived exon regions of the two variants implies that the PFP-p gene represented by the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene form characterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisation analysis indicates that PFP-p is differentially expressed at different stages of sucrose accumulation. PFP-p expression is higher in the immature culm tissue of sugarcane and low in the mature culm, which suggests that PFP-p is highly regulated during maturation. It is hypothesised that the PFP-p gene underwent duplication and that one gene form was subject to accumulative mutations evolving into a pseudogene. On the basis of present results, it can be suggested that future genetic manipulation of PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bp fragment. / AFRIKAANSE OPSOMMING: Suikerriet, 'n komplekse poliploïede organisme, kan teoreties tot 12 allele vir 'n spesifieke geen by 'n enkele lokus bevat. Die aantal allele en hul mate van variasie is veral van belang, weens die potensiaal vir die benutting van hierdie genetiese variasie deur teëling. Alleelvariasie het ook implikasies vir die manipulering van geenfunksie via genetiese inginieurswese. Pirofosfaat-afhanklike fosfofruktokinase (PFP) is 'n belangrike regulatoriese ensiem vir sukrose biosintese en kan geteiken word vir genetiese manipulasie met die oog op verhoogde sukroseproduksie in suikerriet. Die ensiem bestaan uit 'n regulatoriese (alfa) en katalitiese (beta) proteïen subeenheid wat deur die PFP-a en PFP-~ gene respektiewelik gekodeer word. Die PFP-~ geen, wat in ander plantspesies as 'n enkellokusgeen aangedui is, is in hierdie studie gebruik as 'n model vir alleelvariasie in suikerriet. Die twee hoofroetes van ondersoek wat gevolg is, behels genoom- en uitdrukkingsanalises om die geen verder te karakteriseer. Eksons 10 tot 12 van die suikerriet PFP-~ geen is geamplifiseer met behulp van die Polimerase Ketting Reaksie (PKR), bemiddel deur die gebruik van spesifieke voorvoerders wat voorheen ontwerp was vanaf gekonserveerde areas van die PFP-~ geen uit verskillende plantbronne. Twee PKR produkte, genoem PFP-B1/B81250bPen PFP-B1/B81100bPrespektiewelik, is deur die kommersiële kultivars N19 en N21 opgelewer. Verskeie fragmentklone is gekonstrueer en hul DNA basisvolgorde is bepaal. Soektogte in internasionale databasisse het bevestig dat beide amplikons PFP-~ was. Vergelykende DNA basisvolgorde analise het aangedui dat PFP-B1/B81250bP en PFP-B1/B81100bP fragmente swak homologie toon, terwyl 'n hoër mate van homologie in die oënskynlike ekson areas (77-78%), vergeleke met die intronareas (34-56%) gevind is. Alhoewel klein basisvolgordeverskille opgemerk is binne die amplikonpopulasies, was dit duidelik dat twee hoofvariante van die PFP-~ geen in suikerriet teenwoordig is. Southern hibridisasie analisie het 'n eenvoudige band patroon vir PFP-~ onthul. Daar is ook DNA polimorfismes vir die genoomstreke wat met die PFP-B1/B81250bPen PFP-B1/B81100bPfragmente ooreenstem. Vorige bewyse het aangetoon dat beide variante ook in die voorvaderlike suikerriet kiemplasma voorkom en dat dieselfde vlak van heterosigositeit gehandhaaf word. Die voorkoms van beide geenvorme in die voorvaderlike asook kommersiële kiemplasmas stel voor dat hierdie twee variante miskien nie segregeer nie. Hierdie teorie, tesame met die eenvoudige Southern hibridisasiepatroon vir PFP-p, lei tot die hipotese dat die twee geen vorme by verskillende lokusse in die suikerrietgenoom voorkom, en dat hierdie lokusse nou gekoppel mag wees op dieselfde chromosoom. Die uitdrukking van die variante is ondersoek gedurende verskillende stadia van sukrose akkumulasie in die suikerrietstingel deur van Trutranskripsie (TT)-PKR gebruik te maak. 'n Enkele, identiese transkripsie produk is hieruit en uit ander geselekteerde plantweefsels geïsoleer. Dieselfde transkrip is ook verkry vanaf die voorouerlike spesieverteenwoordigers van hedendaagse suikerriet, Saccharum officinarum en Saccharum spontaneum. 'n DNA basisvolgordevergelyking tussen die getranskribeerde produk en die ekson-areas van die twee variante impliseer dat die PFP-p geen verteenwoordig deur die PFP-B1/B81250bPvariant uitgedruk word in suikerriet, terwyl die geen verteenwoordig deur die PFP-B1/B81100bpamplikon stil is. Northern hibridisasie analise toon aan dat PFP-p verskillend uitgedruk word gedurende verskillende stadia van sukrose akkumulasie. PFP-p uitdrukking is hoër in die onvolwasse stamweefsel van suikerriet en laag in die volwasse stam, wat voorstel dat PFP-p hoogs gereguleer word gedurende maturasie. Daar word gehipotetiseer dat die PFP-p geen duplikasie ondergaan het en dat een geenvorm onderworpe was aan akkumulerende mutasies wat deur evolusie tot 'n pseudogeen gelei het. Dit word voorgestel, gebaseer op die huidige resultate, dat toekomstige genetiese manipulasie van die PFP-p geen, die geenvariant gekarakteriseer deur die PFP- B1/B81250bPfragment moet behels.

This thesis examines the interconnection between the in vitro and in vivo phosphorylation of rabbit muscle phosphofructokinase. The first goal of the project was to show whether a novel site of rabbit muscle phosphofructokinase that is subject to in vitro phosphorylation, serine 376, may also become phosphorylated in vivo. Evidence obtained through iron chelate chromatography, amino acid analysis, gas phase sequencing, ammonium sulfate reversed phase high pressure liquid chromatography (HPLC), and matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) spectroscopy of cyanogen bromide digests of the enzyme purified from epinephrine-stimulated rabbit hearts demonstrate the in vivo phosphorylation of serine 376. Parallel experiments with phosphofructokinase isolated from unstimulated rabbit hearts show no detectable phosphorylation of serine 376. The second part of the thesis examines the effects of alterations in experimental conditions on the in vitro phosphorylation of rabbit muscle phosphofructokinase that is catalyzed by the cAMP-dependent protein kinase (cAMP-dPK). Significant phosphorylation of serine 376 takes place in the presence of specific proteins, calmodulin-calcium and troponin C-calcium, that are known to stabilize the catalytically inactive phosphofructokinase dimer. Conditions that stabilize the catalytic activity of phosphofructokinase generally inhibit the in vitro phosphorylation reaction. / Graduation date: 1999

The number of allosteric sites and active sites in phosphofructokinase from Bacillus stearothermophilus create an intricate network of communication within the enzyme. With thermodynamic linkage analysis, the overall allosteric communication can be quantified. This value, however, represents an average contribution for all the interactions involved. The recent development of a hybrid strategy has allowed for the quantification of single interactions, both heterotropic and homotropic. Focusing on the heterotropic interactions whose inhibition is entropy-driven, residues and regions within the enzyme can now be identified to further characterize each specific interaction using the hybrid strategy. Among the many components of entropy, the hybrid strategy has now allowed for the strategic placement of a reporter of side chain dynamics to identify conformational differences between the four ligand bound enzyme species of a single heterotropic interaction. In this study, a combination of these approaches was used in the methodology including constructing hybrids to isolate a single heterotropic interaction along with single tryptophan reporter. Site directed mutagenesis combined with the hybrid strategy was also implemented to directly assess the role of a single residue in the communication path of a single interaction. The region surrounding the allosteric site with the nearest active site has been implicated to be significant in transmitting the allosteric signal. In addition two single residues, T158 and D59, within this region have been identified to potentially contribute to the inhibition of this same interaction. An additional residue, G184, located outside this local region has also been identified as possibly having a significant role in the transmission of the inhibitory signal of a unique heterotropic interaction. The implications of this study have led to the initial identification of residues involved in the 22A route of allosteric communication of a single active site and allosteric site. This allosteric communication occurs to allow the enzyme to compensate for the binding of both ligands. With the location of these residues implicated to be involved in the communication of this isolated interaction, this compensation is not contained within a confined region but is however felt throughout the single subunit.

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme with an average of one active site and one allosteric site per subunit. BsPFK is inhibited by phosphoenolpyruvate (PEP) and how this inhibitory signal is propagated throughout the enzyme is the main question we address through this investigation. By possessing a total of eight binding sites, a potential for twenty-eight total pair-wise allosteric interactions result within BsPFK, ten of which are unique. Of these ten interactions, four are heterotropic interactions, or interactions between unlike binding sites, while the remaining six interactions are homotropic interactions, or interactions between like binding sites. Thus, to address the question of how BsPFK is inhibited by PEP, each of these ten interactions needs to be quantified and their roles in the inhibition process assessed. In order to quantify the roles of the 10 allosteric interactions, we created, purified and characterized several different hybrid enzymes by using site-directed mutagenesis to reduce the number of native active sites and native allosteric sites to permit the isolation of specific allosteric interaction(s). Through the creation and isolation of 1:3 hybrid enzymes, in which one native active site and one native allosteric site remain, each of the four heterotropic interactions were characterized. Moreover, through the creation and isolation of the 2:2 hybrid enzymes, in which two native active sites and two native allosteric sites remain, characterization of the remaining six homotropic interactions was performed. Utilizing a linked function approach to quantify the heterotropic and homotropic effects for each hybrid enzyme, we determined that 5 to 6 of the ten pair-wise allosteric interactions found in BsPFK are involved in the inhibition process depending upon pH. More importantly however, our data provides definitive results that the traditional two-state models used to describe an allosteric effect are not sufficient to describe the allosteric effect measured for BsPFK. Rather, our results show that the linked function approach is a more appropriate way to unambiguously measure the nature and magnitude of an allosteric effect. Moreover, this approach can also be used to explain the allosteric behavior of a dimeric enzyme.

The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.

Thesis (M.Sc. Biochemistry)--Mahidol University, 1979.

A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.

The breakdown of glucose to pyruvate, known as glycolysis, is a central biochemical pathway, critically important for energy production and biosynthesis. Phosphofructokinase (PFK), the third enzyme in the pathway, is a crucial regulator of glycolytic flux, being the first committed step of glycolysis and modulating entry into the pentose-phosphate-pathway. Alterations in PFK activity have been implicated in many neurological conditions, including Tarui's disease, epilepsy, Alzheimer's disease, Down's syndrome, and cancers. There are three isoforms of human PFK; it is assumed that these evolved to fulfil specific metabolic niches both within cells and between tissue types. However, the differences between isoforms have never been systematically compared. Understanding these differences is an essential prerequisite for developing novel therapeutic agents targeting human PFK. Trypanosomatid parasites are a major global cause of neurological morbidity and mortality. Neglected tropical diseases caused by trypanosomatid parasites include African Sleeping Sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). There is increasing interest in targeting the metabolic enzymes of these parasites, including PFK, which greatly differ from mammalian counterparts. This thesis describes biochemical, bio-physical, and bio-informatic studies on the three human PFK isoforms (PFK-M, PFK-L, and PFK-P), expressed in S. cerevisiae. Biophysical studies showed that the active conformation was tetrameric, with activity regulated by time and concentration dependent dissociation into smaller inactive species. The propensity to dissociate differed between isoforms, with PFK-M being most stable and PFK-P least stable. Dissociation was synergistically slowed by the addition of substrates and reducing agents, indicating different mechanisms of action. Kinetic studies were performed with respect to both substrates (ATP and F6P) in the presence of natural metabolites hypothesised to act as modulators of enzyme activity. Each isoform conformed to an allosteric sigmoidal kinetic model and had differing kinetic properties, with PFK-M being the most active and PFK-P the least active. ATP was found to act as both substrate and allosteric inhibitor, with activity showing a biphasic response to ATP concentration. Each isoform showed different susceptibilities to both ATP inhibition and regulation by allosteric modulators. The reverse reaction was shown to be possible under certain conditions. Bio-informatic data on intra-cellular and inter-cellular locations were determined using the Human Protein Atlas and the FANTOM5 datasets. PFK-M localises to the cytosol and may co-localise with endoplasmic reticulum; PFK-L associates with nucleoli and mitochondria; and PFK-P is cytosolic. Splice variants were not shown to be physiologically significant. Each isoform had different tissue expression levels, with overall PFK expression varying by tissue type. PFK-P was the principal isoform in cancers, whereas PFK-L was dominantly expressed in immune cells. Activated macrophages switched rapidly from PFK-L to PFK-P. PFK-M and PFK-P were the dominant isoforms in the brain, although there were differences between brain areas. Neurons expressed less PFK than astrocytes, in keeping with the lactate shuttle theory. PFK from each of the three main pathological trypanosomatid species were compared (T. brucei, TbPFK; T. cruzi, TcPFK; L. infantum, LmPFK); expressed in E. coli. Biophysical analysis showed each PFK to be tetrameric; no evidence of time or concentration dependent dissociation or inactivation was found. Kinetic properties differed between isoforms, with TcPFK being most active and LmPFK being least active. LmPFK was very poorly active with regard to F6P titrations unless AMP was present. No other modulators were shown to affect activity, although GTP was an alternate substrate. The reverse reaction was shown to be possible and may be compatible with physiological concentrations of ADP and F16BP in the trypanosomatid glycosome.

It has recently been established that the activity of crystalline muscle phosphoglucomutase can be greatly stimulated by preincubation of the enzyme with a Mg++-imidazole complex. This observation has aroused interest in the physiological significance of such a system in the possible cellular control of phosphoglucomutase activity. The present study constitutes, in part, an investigation of the properties of phosphoglucomutase from brain tissue. A procedure for the purification of phosphoglucomutase from beef brain is described. The brain enzyme appears to be similar to that from skeletal muscle. Evidence is also presented which indicates that the "activation" produced by Mg++-imidazole is probably of no physiological importance in brain. This observation is consistent with the more recent reports that the phosphoglucomutase reaction is likely not involved in cellular regulatory mechanisms. It is well established that phosphofructokinase is intimately involved in the cellular regulation of glycolysis and the citric acid cycle. Control mechanisms of the phosphofructokinase reaction in mammalian tissues have been postulated on the basis of the complex kinetics of the enzyme. In yeast, however, two enzymatically interconvertible forms of the enzyme have been reported. Preliminary experiments in this study failed to demonstrate a phosphofructokinase system in brain similar to that found in yeast. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate