Part I of this thesis describes the digestion of bakers' yeast s-RNA at 0° and at room temperature in the presence of various concentrations of magnesium ions and at several different s-RNA : enzyme ratios (w/w). Digestion of s-RNA with pancreatic ribonuclease (ratio, 2000:1, (w/w) ) in the presence of 0. 2 M magnesium and at 0°, followed by chromatographic analysis, indicated that about 75% of the ultraviolet absorbing material was of medium to high molecular weight. Analysis of the nucleotide composition of the fraction containing the intermediate size oligonucleotides showed that fraction to be enriched in the odd bases ψ, T, 1-methylG, N, N-dimethylG and several other unidentified nucleotides. This result was incompatible with the 'hairpin' models for s-RNA, proposed by McCully and Cantoni (1962). The presence of spermine in the digestion mixture clearly masked the catalytic action of pancreatic ribonuclease, albeit to a lesser extent than magnesium ions, contrary to a report by Thomas and Hubst (1963) in their study of the affect of spermine on the catalytic activity of E. coli ribonuclease toward E. coli ribosomal RNA. The studies described in Part II of this thesis are concerned with the fractionation of mixed s-RNA from yeast. Part II-A describes a new chromatographic material consisting of a bifunctional mercuri-dioxane derivative attached through a thiol group to the cellulose matrix. A previous report (Eldjarn and Jellum, 1963) had shown that HS-proteins could be fractionated on an organomercurial-cellulose (Material I) derived from aminoethyl-cellulose. The studies reported, here suggest that ¹⁴C-cysteinyl-s-RNA cannot be retained on Material I because the salt concentration required to overcome its ion-exchange properties preclude any interaction between the mercury of Material I and the sulfhydryl group of ¹⁴C-cysteinyl-s-RNA. The present study describes how this disadvantage was overcome by preparing an organomercurial-cellulose devoid of ion-exchange properties (Material II). Preliminary studies showed that Material II can retain the radioactivity associated with ¹⁴C-cysteinyl-s-RNA. These studies suggested that Material II might fractionate nucleic acids according to their base composition. Part II-B describes some of the attempts to fractionate yeast s-RNA by column partition chromatography. In general, resolution of acceptor activities of s-RNA, as well as the recovery of biological activity, was poor. Part II-C describes some of the author's contributions to the studies carried out in this laboratory on the fractionation of yeast s-RNA on another new chromatographic material, benzoylated-DEAE-cellulose (Material III). Certain variables (such as, magnesium ion concentration and pH), which can be manipulated during rechromatography of s-RNA on Material III, are discussed in light of the chromatographic behaviour of glycine and other acceptor RNA's. The forces responsible for this fractionation are also discussed. Part II-D describes a chemical procedure for the isolation of glycine s-RNA which, when combined with the chromatographic procedures described in Part II-C, afforded glycine-specific s-RNA, in high purity. Observations on the effect of magnesium ion during enzymatic synthesis of glycyl-s-RNA are discussed. The advantages and possible consequences of using purified aminoacyl-s-RNA synthetase enzymes for the preparation of the aminoacyl-s-RNA's are also discussed. The need to prepare large quantities of aminoacyl-s-RNA synthetases derives from their indispensable role in the development of chemical methods for purifying amino acid-specific s-RNA's (see Part II-D). Grinding of yeast cells with glass beads, which is the usual method employed for disrupting yeast cells, was found to be inadequate. Lysis of yeast cells with toluene at 37° was found to be a partial solution to this problem. Although previous studies showed that most of the synthetase enzymes were destroyed by this treatment (von Tigerstrom and Tener, 1967), investigations described in Part III of this thesis, show that most of the synthetase activities can be preserved by controlling the pH of the toluene-yeast mixture. However, there were still several synthetase activities missing in the cell-free extracts prepared by the warm toluene method. Preparation of large quantities of yeast cell-free extract containing all of the aminoacyl-s-RNA synthetases was finally achieved by treatment of the yeast cells with toluene containing excess dry ice followed by an incubation period at 3-5°. Partial purification of the aminoacyl-s-RNA synthetases was achieved by chromatographing the cell-free preparation on hydroxyl-apatite. The presence of 40% glycerol in the eluting buffers was found to be essential for the preservation of most of the synthetase activities. When the cell-free preparations were chromatographed on hydroxy-apatite, most of the protein, but only a few of the synthetases were eluted by a linear gradient of phosphate buffer to 0.20 M. Most of the synthetases were eluted only when the column was washed with ammonium sulfate. The significance of this observation is discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Interest in brain catecholamines has grown considerably in the last few years in view of their possible role as neurotransmitters. This investigation deals primarily with the enzyme tyrosine hydroxylase which is thought to be the rate limiting step in catecholamine synthesis. Using tyrosine hydroxylase measurements and catecholamine depletion techniques,attempts were made to determine the site of increased synthesis of catecholamines in animals exposed to cold. Brain, heart and spleen do not appear to be the organs involved in this change. Adrenals may be of significance but the results were suggestive rather than conclusive. Tyrosine hydroxylase distribution in brain was determined in various regions of rat, rabbit and cat brain, and activity was shown to be highest in the caudate, septal area, nucleus accombens, and anterior perforating substance, with much lower activities in other regions such as hippocampus, amygdala, hypothalamus, thalamus, cortex, cerebellum and brain stem. Using these distribution studies as indications of normal tyrosine hydroxylase activity in areas of rat brain, and electrolytic lesion techniques, studies were carried out to determine noradrenergic and dopanergic pathways in brain. Catecholamine fibers from their origin in the midbrain were traced in the midbrain and diencephalon to the caudate and septal area, and the relative positions of each group of fibers determined along their course. / Medicine, Faculty of / Graduate

Although the quantity of glycogen in nervous tissue is small, in recent years it has been found that there is a significantly high turnover rate of this polysaccharide. Glycogen is present in sympathetic ganglia, midbrain structures, white matter, and synaptic regions. The enzymes of glycogen synthesis and breakdown exist in brain in high concentrations, which suggests that glycogenolysis may be coupled in some way to the energy-requiring processes concerned with electrical activity. In this thesis, the occurrence and activity of phosphorylase a, phosphorylase b kinase and phosphofructokinase were determined in peripheral and central nerve tissue, an attempt to purify phosphorylase b kinase was made, and the mechanisms controlling the activity of the brain kinase were studied. These experiments were carried out in an effort to learn more about the importance of glycogenolysis in the nervous system and the factors which control it. The results of a survey of dog nerve tissue and some experiments on rabbit tissue indicated that the enzyme levels in brain, superior cervical and stellate ganglia were comparable to those in heart. Other peripheral nerve tissue has approximately one-tenth the level found in brain. Heavily myelinated axonal tissue contained higher phosphorylase activity and much higher kinase activity than axons with relatively little myelination. It is suggested that glycogenolysis is likely to be concerned with synaptic transmission and Schwann cell metabolism. Attempts to purify one of the controlling enzymes in glycogenolysis, phosphorylase b kinase, from brain tissue were hampered by the instability of the enzyme and the consequent loss of activity that occurred at each purification step. A modest 3.0 to 3.5-fold purification was achieved by differential ultracentrifugation, freezing and thawing of the 100,000 x g precipitate, and a 0-35 per cent ammonium sulfate fractionation at pH 5.7. The enzyme exhibited increased stability when stored in 50 per cent glycerol at -18°, especially when it was present in a more purified state, but other attempts to increase the stability of the kinase met with little success. The maximum activity of brain phosphorylase b kinase occurred between pH 8.75 and 8.9, and a significant activity was noted at pH 7.0, which is somewhat different from that found in other tissues. However, the brain kinase is activated by mechanisms similar to those found for skeletal and cardiac muscle. The activity at pH 6.8 is increased by preincubation with Ca⁺⁺, and further increased by the addition of rabbit skeletal muscle calcium-activating factor. This is partially prevented by beef heart calcium inhibitory factor. ATP-Mg⁺⁺ and cyclic AMP also cause activation of the brain kinase. The total activation of the brain enzyme appeared much lower than that found in skeletal or cardiac muscle due to the already high activity at pH 7.0. Experiments designed to produce an accurate value for the pH 6.8/8.2 ratios of kinase activity utilized freezing of mouse brain in vivo in liquid Freon cooled to its freezing point. The value of 0.44 obtained from these procedures was similar to, or slightly higher than, the ratios in rabbit and beef brain. The kinase was not inactivated by room temperature incubation for two hours, although similar treatment would convert phosphorylase to its inactive form. The possibility remains that the pH 7.0 activity is artificially high, assuming the freezing techniques used are not fast enough. Otherwise, the high enzymatic activity may be due to the continuous electrical activity present in the brain. The possibility that glycogen catabolism is a more important source of energy in the nervous system than previously thought is supported by the high levels of enzymatic activity and similar phosphorylase b kinase control mechanisms to those in other tissues. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

The mechanism of rabbit skeletal muscle phosphoglucomutase (EC.2.7.5.1) has been investigated using fluorinated and deoxygenated substrate analogues. Each of the analogues in which the non-acceptor hydroxyls are replaced by fluorine or hydrogen are substrates of the enzyme. The kinetic constants of these substrates are reported. The rate of the mutase reaction of each substrate analogue in the presence of glucose 1,6-diphosphate is the same as that of the half reaction involving production of the fluorinated and deoxygenated glucose 1,6-diphosphate species. The exceptions are 3-fluoro- and 3-deoxy-glucose 1-phosphate, in which cases the rates of the half reactions are 8 times that of the overall mutase reaction. The Km of 3-fluoro-glucose 1,6-diphosphate is approximately 90 times that of glucose 1,6-diphosphate and the other deoxy and fluoro analogues. The inhibition of phosphoglucomutase by fluorinated and deoxygenated substrate analogues has been investigated. The synthesis of a series of novel disubstituted inhibitors (based on glucose 1-phosphate) in which the C-6 hydroxyl is replaced by fluorine and a sugar ring hydroxyl is replaced by either hydrogen or fluorine is described. The inhibition constants show that the hydroxyl distal to the acceptor hydroxyl is most important in the formation of a strong enzyme-inhibitor complex. The synthesis is described of three phosphorofluoridate analogues of glucose phosphate substrates. These analogues were found to only weakly inhibit phosphoglucomutase. No evidence of any phosphoryl transfer between the phosphoenzyme and the phosphorofluoridate analogues could be detected. Thus phosphoglucomutase has a strict requirement for a doubly negatively charged substrate phosphate group. The interaction of phosphoglucomutase with fluorinated substrates and inhibitors has been investigated by ¹⁹Fnmr. Large downfield changes in the chemical shifts of the inhibitors 6-fluoro-glucose 1-phosphate and α-glucosyl fluoride 6-phosphate were found to accompany binding to the phosphoenzyme. The effects of the binding of activating and non-activating metal ions on these spectra were investigated. The different effects observed may be directly related to the chemical basis for the metal induced activation of the enzyme. ¹⁹Fnmr data consistent with a 10² to 10³ fold increase in the tenacity with which phosphoglucomutase binds substrates and inhibitors in the presence of Li⁺ were observed in the spectra of the phosphoenzyme with difluorinated glucose 1-phosphate inhibitors. Two enzyme bound species were detected in the ¹⁹Fnmr spectra of the complexes formed by reaction of the Cd²+ phosphoenzyme with 2- and 3-fluoro-glucose phosphates. These species are tentatively assigned as the fluoro-glucose 1,6-diphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of 4-fluoro-glucose phosphates. The environment of each substrate glucose hydroxyl in the active site was probed using ¹⁹Fnmr and the fluorinated glucose phosphate substrates. Data inconsistent with a minimal motion type of mechanism (W.J. Ray, A.S. Mildvan & J.W. Long, Biochemistry 1973,12, 3124) were obtained. The results of the nmr and kinetic studies are consistent with an exchange type of mechanism in which the C-3 hydroxyl plays an important role in the reorientation of the glucose 1,6-diphosphate. The data also suggest that there are two distinct glucose binding sites, one for each substrate and glucose 1,6-diphosphate bound in the same mode. / Science, Faculty of / Chemistry, Department of / Graduate

Magister Scientiae - MSc / Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50°C. AXE activity was stable for at least 1.5 hours between 30°C and 40°C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30°C-40°C suggests potential for industrial applications that utilise mesophilic fermentations. / South Africa

Magister Scientiae - MSc / The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (“ubiquitinated”) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called “ubiquitin-like modifiers”, which consist of a single domain that structurally resembles ubiquitin, and “ubiquitin-domain” proteins, which are multi-domain proteins, which include domains that resemble ubiquitin. This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained. Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb. / South Africa

Alternating (CG) sequences form an unusual conformation in the presence of cobalt hexamine. The oligomer, BZ-IV, containing a (CG)4 run (BZ-IV sequence: 5'TCGACGCGCGCGATCAGTCA- 3') was inserted at the Sal I site of the Escherichia coli pGEM-5zf(+) plasmid producing the plasmid pCW001. Hinf I digestion of pCW001 produced a 367 base pair (bp) fragment containing the BZ-IV insert. For controls, the 452 bp Hinf I fragment from the pCW001 plasmid and the 347 bp Hinf I fragment from the pGEM plasmid were used. Digestion studies were performed using the restriction enzymes Bgl I, EcoRV, Hha I, Mbo I, Not I, Pst I, and Taq I and methylation studies were performed using dam methylase. Data were obtained by beta scanning or ethidium bromide staining the polyacrylamide gels of the digestion or methylation products. The results show that in the presence of 100 uM cobalt hexamine, in which BZ-IV takes on a non-B-Z-structure, the enzyme's ability to react and cleave its recognition site is enhanced.

Bibliography: pages 79-86. / Flavour in wines is perhaps the most important factor affecting wine quality, with monoterpenoids being among the compounds contributing to flavour. In grapes there exists glycosidically-bound forms of these monoterpenes, representing a latent source of aroma. This project is a study on the ability of different enzymes to release these monoterpenes in Muscat d'Alexandrie and Weisser Riesling grapes. Different commercial enzyme preparations were characterised with respect to their catalytic properties and fractionated by ion exchange chromatography in the most active fractions. Amongst those commercial preparations tested, Rohapect C was found to be the most effective at conditions prevalent during wine making. These purified fractions were added to the above mentioned grapes to determine their effect on the release of different monoterpenes. Even though the enzymes were active on synthetic substrates, limited release of terpenes from must could be detected. Possible causes for this apparent inability to release monoterpenes were investigated. Product inhibition due to the presence of high concentrations of glucose in the must appeared to be the main cause of limited enzyme activity. In an attempt to overcome this problem, glucose in the above mentioned cultivars was oxidised to gluconic acid by glucose oxidase, prior to attempting monoterpene release by Rohapect C. Although no marked increase in the release of total terpenes occured, a significant increase in the concentrations of some individual terpenes could be observed. The effect of this on wine quality remains to be ascertained.

Purification of the phosphoroclastic system of Escherichia coli was undertaken. A 4-fold purification of the crude extract was obtained using heat precipitation and protamine sulfate. Some purification of Knappe's A fraction was obtained with ammonium sulfate and acetone. Evidence was obtained for the existence of two factors in the A fraction. Other purification techniques gave little success. Good evidence was obtained for the involvement of phosphotrans-acetylase in the reaction sequence. Phosphotransacetylase was purified 750-fold and characterized. The reversibility of the reaction was studied with carbon 14. Formate fixed readily into pyruvate only when pyruvate was present. Acetyl coenzyme A fixed into pyruvate also but to a much smaller degree. Better fixation without pyruvate was obtained after prior incubation and consumption of pyruvate. The dilution effect, inhibitors, effect of light, and a possible role of coenzyme B_12 were investigated. By using a deuterium label, it was shown that a hydrogen does not shift directly from the methyl group of pyruvate to formate in the reaction mechanism.

Serine transhydroxymethylase was purified one thousand-fold from bovine brain and one hundred and twenty-fold from bovine liver. Both enzymes were yellow due to the presence of the coenzyme pyridoxal 5'-phosphate. Polyacrylamide gel electrophoresis of the liver enzyme showed one major band containing 95% of the protein, while in the case of the brain enzyme two major bands of nearly equal intensity were observable. The specific activity of the crude liver enzyme was about sixty times higher than that of brain. The liver enzyme was also more heat stable than the brain enzyme. The purified enzymes exhibited L-allothreonine and L-threonine aldolase activities besides serine transhydroxymethylase activity. Electro focusing established the isoelectric point of liver enzyme at 8.75 and a value of 8.90 was obtained from the brain enzyme. The molecular weight of the brain enzyme was found to be 229,000 ± 20,000 while the liver enzyme had a value of 245,000 ± 20,000. pH optima of the enzymes were around 8.0. A similarity in antigenic properties was established for the enzymes from both tissues.