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The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.

M.Sc. / Aerosol samples were collected at stations using simple filter unit, loaded with a 4 7 mm diameter pore size Nuclepore membrane filter. The sampling duration was 24 hours for each sample, with the total of 11 samples: seven for Thohoyandou and four for Bethlehem. The samples were analyzed with energy dispersive X-ray fluorescence spectrometry for up to 20 elements. Comparison for elemental concentrations of the data showed that the samples fall into three clusters on average (major: Ca, Cl, Si. S, Fe and AI; intermediate: K, Ti, Pb, Brand Zn; minor: Mn, Cr, As, Sr, Ni and Cu), with the samples collected from Bethlehem having high concentrations in all clusters. High concentrations in the samples collected on Bethlehem were attributed to entrainement of dust particles during the dry winter period. The results also show that the elements are associated with anthropogenic emissions. Therefore, the sampling station at Thohoyandou is considered as a background station due to the low aerosol concentration. The aerosol concentration levels at Thohoyandou can be attributed to biogenic sources because of the large presence of large forest areas in the region and in the vicinity of the sampling location. The samples collected in both regions reveal highly enriched sulphur, chlorine, zinc and lead. It is clearly proved that these elements come from local soil. Chromium and copper were both slightly enriched in Thohoyandou, but the concentration data for these two elements are not available. However it is supposed that these elements come from local soil as well. In another aerosol analysis, passive (diffusive) samplers were used to measure concentration levels for sulphur dioxide and nitrogen dioxide. The measurement campaign was carried out from Soweto in Johannesburg and Bohlokong in Bethlehem with the campaign lasting for two months during the w~nter season. A very clear result derived for sulphur dioxide was the dominating source contribution from use of coal for heating and cooking in both areas. For nitrogen dioxide, it was found that contribution from traffic in highly populated areas and from industrial activities in the neighbourhood of the two areas was the source. The role played by wood burning, is also another additional source used for domestic heating and even cooking.

One problem of intensive fish culture systems is the progressive build-up of toxic wastes such as ammonia. The possibility of improving aquaculture water quality using two kinds of zeolite is discussed. Zeolites are alumino-silicates whose framework allows them to exchange cations. Ion exchange has been demonstrated to be competitive with other methods of ammonia removal due to the high selectivity for ammonia exhibited by zeolite materials. In this study an unknown Zambian zeolite (identified as laumontite by X-ray diffraction techniques) and Pratley clinoptilolite (a South African zeolite) were tested under laboratory conditions and in a fresh water recirculating system. Ammonia cation exchange capacities (CEC) and suitable application rates for efficient water treatment were determined using the batch and column ion exchange procedures. Estimated ammonia uptake, the most important criterion used to assess performance of zeolite filters was strongly influenced by zeolite type, particle size, pre-treatment, regeneration and ion exchange method used. Statistical analysis showed significant differences in average ammonia CEC values between clinoptilolite (14.94 mg g⁻¹) and laumontite (2.77 mg g⁻¹), with the former displaying a higher Na⁺ ® NH₄⁺ exchange rate especially in the early reaction stages. This difference accords with the higher purity of clinoptilolite, 47% as opposed to 4.7% for laumontite, which makes it a better zeolite for ammonium removal. CEC increased linearly as particle size of the clinoptilolite was reduced resulting in a linear regression model (y = 18.29 – 3.704 x; r² = 74%). Pre-treatment of clinoptilolite using 1N NaCl significantly improved the ammonia CEC of clinoptilolite. Overall performance of both the batch and column methods achieved after regeneration (18.3 mg g⁻¹) was 25% higher than the estimated CEC values (13.0 mg g⁻¹) for the unregenerated samples of clinoptilolite. Comparison of CEC estimates using Pratley clinoptilolite, showed that average batch CEC estimates were significantly lower than the column method estimates. The average ammonia CEC values estimated in a fresh water recirculating system (5.80 mg g⁻¹ and 4.12 mg g⁻¹ for the 0.7-1.0 and 1.0-1.4 mm particle sizes, respectively) were significantly lower than the column and batch estimates for the same particle sizes (P < 0.05). Some nitrite (NO₂) and nitrate (NO3) build up was experienced probably due to the growth of autotrophs in the filters. Mass balance of nitrogen (N) for the three treatments of the fish trial (0.7-1.0 mm, 1.0-1.4 mm and the control treatment that had no zeolite in the filter) indicated that less that 10% of the N was retained for growth. It was found that 60% of the NH₄-N present associated with the soluble N was available for absorption by the zeolite filter or biological nitrification and that a total of approximately 22% of NH₄-N available was absorbed by clinoptilolite. The results indicate that the rate of nitrification can be deductively estimated by allowing a zeolite filter to become a biological filter. It is concluded that water treatment by ion exchange using natural zeolites, provides a reliable and efficient method for ammonia removal and appears to be a viable supplementary water treatment method for fresh water systems.

The radioactive isotope K⁴² was used to measure the rate of potassium exchange in muscle from 129 strain mice. The results followed an unique course if plotted as K⁴² uptake versus (external potassium concentration ∙ time) [superscript ½], and corresponded to the result predicted for K⁴² uptake mediated by an ion-exchange compartment in the muscle. Variations in external potassium concentration did not affect the uptake rate if plotted this way, but sodium ion exerted some effect on the rate. Dystrophic mouse tissue accumulated K⁴² more rapidly than did normal tissue, and the effect of varying the external potassium concentration did not alter this rate. The effects of sodium variation were more pronounced than in normal tissue. Inulin space of muscle was measured in vivo as well as in vitro, to enable a correction for K⁴² in the extracellular space to be made. The inulin space was found to decrease with increasing muscle size, and this was thought to be related to the development of the muscle. Dystrophic muscle exhibited more of a dependance of inulin space on muscle size than did normal muscle. The suggestion was made that the dystrophic muscle membrane might be abnormally permeable to inulin. Muscles were excised and assayed by flame photometry for sodium and potassium content. They were assayed when freshly excised, and also following incubation in a variant of Locke’s solution. The muscle cations were stable for the first two hours of incubation, but after this time, intracellular sodium rose and potassium fell. Fresh dystrophic mouse muscle had lower potassium and higher sodium content than normal fresh muscle. The cation changes following incubation resembled those found for normal muscle. The changes in intracellular cations were correlated with the K⁴² uptake results, and discussed in some detail. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Previous studies of selective reabsorption in insect excretory system have concentrated almost exclusively on the rectum, while the role of the ileum has been assumed to be minor. The properties and control of solute and fluid transport in two segments of the hindgut, the ileum and rectum, from the desert locust (Schistocerca gregaria) have been studied and compared in vitro using everted sac and flat sheet preparations. Everted sacs of locust ileum transported fluid from the lumen side to hemocoel side over a 5 h period at near constant rates of 3.0 to 3.5 μL-h⁻¹-ileum⁻¹ and tissue volume did not change. Inhibition by azide indicated metabolic dependence of fluid transport. Fluid absorption occurred against osmotic concentration differences of up to 600 mosmol. Fluid transport was stimulated by cAMP, both nervous and glandular lobes of corpus cardiacum (CC), and fifth ventral ganglia (VG) in a dose-dependent manner. All stimulants caused ilea to absorb against larger osmotic concentration differences than unstimulated sacs. The ileal absorbate remained hyperosmotic to the luminal saline under all conditions and stimulants increased absorbate osmolality. Unstimulated fluid transport was supported at 50% of control levels by any one of Na+, K+, or CI⁻. Stimulation of fluid transport by CC or VG was dependent on CI⁻ and maximal stimulation occurred when the Na+:K+ ratio was 1:1. Cyclic AMP, CC and VG all stimulated Na+,K+ and CI⁻ absorption across everted ileal sacs. This is the first direct demonstration that Na+ reabsorption is controlled in insect excretory systems. Stimulation resulted in a decrease in absorbate HCO₃⁻ levels and pH concurrently with an increase in absorbate CI⁻ levels. Stimulation of fluid transport was associated with a 3-fold increase in transepithelial potential (hemocoel negative) suggesting stimulation of electrogenic anion (CI⁻) movement to the hemocoel. Net Na+ absorption occurs largely by electroneutral active transport. NH₄+/Na+ exchange may account for one-third of stimulated net Na+ flux. Extracts from both CC and VG stimulated fluid, K+, and CI⁻ transport across everted rectal sacs, but only a small stimulation of Na+ flux was observed which was an order of magnitude less than that observed for stimulated ilea (0.4 versus 5.1 μequiv-h⁻¹ -cm⁻²). Unlike the rectum, the ileum did not transport proline transepithelially and ileal fluid transport was not stimulated by increasing concentrations of proline in the bathing saline. Rectal fluid transport was stimulated 50% by increasing external proline concentration from 1 to 80 mM. Stimulation of rectal fluid transport by proline also occurred in the absence of Na+, K+, and CI⁻ and occurred against larger osmotic concentration differences. These results are consistent with previous reports of a high capacity transport system for proline in locust rectum. The presence of anion-stimulated ATPase and Na+,K+-ATPase in locust hindgut was also investigated. Anion-stimulated ATPase activities were observed in microsomal fractions of both rectum and ileum. Microsomal fractions from both tissues had enriched specific activities of several plasma membrane marker enzymes and decreased activities of two mitochondrial markers as compared to homogenate enzyme activities. Na+,K+-ATPase activity was 20-fold higher in the rectum than in the ileum, associated with the greater development of the basolateral membrane in the rectum. Overall the results suggest that ion and fluid reabsorption in the locust ileum is much more important in the excretory process than previously supposed. Moreover, this reabsorption was shown to be under neuroendocrine control. / Science, Faculty of / Zoology, Department of / Graduate

The cellular mechanism and regulation of KC1 reabsorption across the rectum of the desert locust Schistocerca gregaria has been studied using tracer fluxes, ion-sensitive microelectrodes, and electrophysiological techniques. Serosal addition of 1 mM cAMP stimulates transepithelial short- circuit current (I[sub=SC]) and net Cl absorption (J[sub=net;sup=Cl] ) 10-fold, increases transepithelial potential (V[sub=t]) 4-fold, and reduces transepithelial resistance (R[sub=T]) by 40-65%. The properties of locust Cl transport are not consistent with NaCl cotransport models proposed in other epithelia: i) Cl is absorbed from nominally Na-free saline, ii) there is no correlation between trace amounts of Na contamination and the rate of Cl transport, iii) exposure to cAMP increases ³⁶Cl influx across the apical border into rectal tissue without affecting ²²Na influx, iv) Cl-dependent I[sub=SC] is not inhibited by 1 mM ouabain (2 h) or 1 mN furosemide (1 h), v) J[sub=net;sub=Cl] is not affected when the apical Na electrochemical gradient is reduced by 85%, and vi) there is no relationship between Na and Cl net electrochemical gradients across the apical membrane. Cl/HCO₃ exchange is also unlikely since i) Cl-transport is electrogenic, ii) J[sub=net;sub=Cl] is insensitive to CO₂⁻ and HCO₃⁻ removal, and iii) Cl-dependent I[sub=SC] is not inhibited by 1 mM SITS or 1 mM acetazolamide after 1 h exposure. The cAMP-stimulated system is Cl-selective: Cl >> Br >> I,F,SCN,P0₄,SO₄.C₂H₃O₂,urate. The halide sequence suggests a site having high field strength. Cl-dependent I[sub=SC] is inhibited by low mucosal pH and high osmotic pressure. J[sub=net;sub=Cl] obeys Michaelis-Menten-type kinetics. Mucosal K increases both the K[sub=m] and V[sub=max] of transepithelial Cl absorption (K[sub=a] = 5.3 mM K). The active step in J[sub=net;sub=Cl] is at the apical membrane because net entry of Cl occurs against a large, unfavourable electrochemical gradient. Serosal cAMP and mucosal K directly stimulate the active step since both of these agents cause simultaneous increases in J[sub=net;sub=Cl] and the electrochemical potential opposing CI entry. Passive K transport in the mucosa-to-serosa direction is favoured across apical and basal membranes. Most K absorption (~84%) is electrically coupled to active CI transport under open-circuit conditions, however a small active component is apparent during exposure to cAMP. The response of V[sub=T] to transepithelial salt gradients depends strongly on the direction of the gradients, suggesting that locust rectum is a "tight" epithelium. Intracellular current and fluorescent dye injections reveal strong coupling between rectal cells. Flat-sheet cable analysis indicates that locust rectum becomes "tighter" during cAMP exposure, when transcellular conductance increases from 60 to 95% of the total tissue conductance. cAMP increases apical membrane K conductance and basal membrane CI conductance. K permeability is inhibited by high (physiological) K and osmotic concentrations. The driving force of CI transport is calculated by two independent methods and the results are interpreted in terms of an equivalent electrical circuit model for KCl reabsorption across locust rectum. / Science, Faculty of / Zoology, Department of / Graduate

D.Sc. / The ion exchange properties of a number of synthetic inorganic ion exchangers and the natural zeolite, clinoptilolite, were evaluated with respect to the elements Na, Cs, Sr, Ba, Ce(III), Eu, Zr, U(VI), Np(V), PU(IV) and Am(III). The role of the structure of crystalline exchangers in determining the selectivity thereof, especially in the case of clinoptilolite, was also investigated. Hydrated antimony pentoxide and antimonic acid showed high sorption of a number of ions over a wide pH range (2 - 10). The selectivity order of hydrous oxides for the actinides was as follows: PU(IV) > Am(III) > U(VI) > Np(V). Titanium phosphate, zirconium phosphosilicate and sodium titanate, however, preferred U to Am. To predict the selectivity order of crystalline inorganic ion exchangers, the Eisenman theory could be used, but other factors such as ion sieve effects had to be taken into account. In such cases the parameters contributing to the change in free energy during ion exchange plays a major role. Samples containing clinootilolite from two deposits in South Africa were evaluated with respect to chemical composition, conversion to the sodium, calcium and cesium forms, as well as the sorption of Cs and Sr from various media. Similar trends to those:reported for clinoptilolite from other parts of the world were found. Rietveld refinement of the structure of Cs-exchanged clinoptilolite was carried out using powder diffraction data and the computer program WYRIET. The zeolite was not isolated from the natural matrix, which meant that a number of phases had to be included in the refinement process. The refinement confirmed the...

The object of the research was to study, by means of ion-exchange chromatography, the product mixture resulting from reaction of trans-[Co(en)₂Cl₂]Cl and SCN¬⁻. Cis-and trans-[Co(en)₂(NCS)Cl]Cl had previously been obtained as products of this reaction. However, the possibility of the presence of other products consistent with the nature of the reactants could not be dismissed. These include the dichlora, dithiocyanato, and aqua complexes. The constituents of the solution resulting from the reaction were separated on a column of Dowex 50W-X8 resin by elution with hydrochloric acid. The eluted fractions were evaporated at room temperature in order to obtain crysta1s to be used for infrared spectral analysis and identification. Three primary fractions were eluted from the column. The first, eluted with 0.5 M HCl, was identified as trans-[Co(en)₂(NCS)Cl]Cl by analysis of the infrared spectrum. Two types of crystals were obtained from the second fraction which was eluted with 1 M HCL. The first crystals to form in this solution were orange in color and were identified by examination of the infrared spectrum as cis-[Co(en)₂(NCS)Cl]Cl. The second crystals obtained from the same fraction were shown to be cis-[Co(en)₂(NCS)Cl]Cl. The crystals isolated from the third fraction, eluted with 3 M¬¬ HCl, were also identified as cis [Co(en)₂(NCS) Cl]Cl. On the basis of observations of the column elution and an analysis of the ultraviolet spectra of’ the eluted fractions, it was concluded that band #2 was cis-[Co(en)₂(NCS)Cl] Cl and band #3 was cis-[Co(en)₂(NCS)(H₂0)]Cl₂. Aquation of cis-[Co(en)₂(NCS)Cl]⁺ was observed to occur on the column as well as in the eluted solution. In addition, rechlorination of the aquated compound was found to occur in the eluted fraction so that after elution the second and third fractions were mixtures of the unaquated and aquated complexes. No crystals of dithiocyanato or dichloro compounds were obtained in the separation so that the reaction was demonstrated to be essentially1:1 between the complex and thiocyanate. Trans-[Co(en)₂(NCS)Cl]Cl was found to comprise forty-nine percent of the typical product mixture. Since other products were present only in very low concentrations, the combined cis-[Co(en)₂(NCS)Cl]Cl and cis-[Co(en)₂(NCS)(H₂0)]Cl₂ contents was estimated at roughly fifty mole percent.

As far back as the time of Aristotle sand filters were used to purify water. However it was not until the time of Thompson and Way (1848), two English agricultural chemists, that the idea of actual exchange of ions was proposed. Ion exchange has been investigated extensively, resulting in applications to softening of water, purification of sugar, separation of compounds, recovery of metals, many pharmaceutical separations and many others. It has been proposed that the internal diffusion should be related to the internal structure of the ion exchange resin. The object of this research was to measure the internal diffusion and relate it to the cross-linkage of the resin. It was suggested by Dr. Theodore Vermeulen of the Chemical Engineering Department, University of California. He proposed that the external diffusion could be eliminated by having a high rate of flow in the column. By measuring the width of the zone formed, the rate constant could be found. The rate constant is proportional to the diffusion rate.