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Preparation Of A Flavonol Specific Glucosyltransferase Found In Grapefruit And Site-Directed Mutants For Protein Crystallization, Aaron Birchfield

Electronic Theses and Dissertations

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4^o C was optimal for removing tags from CP3GT. Storage at 4^o C for …

Method Development For Structural Assessment Of Nanolipoprotein Particles With And Without Cross-Linked Lipids, Emma J. Mullen, Wei He, Sean Gilmore, Matthias Frank, Matthew Coleman, Megan Shelby

STAR Program Research Presentations

Membrane proteins make up approximately 30% of the cellular proteome and account for over 60% of pharmaceutical targets.1 Determining the structures of this class of proteins is critical to our understanding of disease states and will advance rational drug design. But membrane proteins have limited solubility, rarely form large crystals that diffract well, and often misfold outside of a bilayer, hindering crystallographic studies.1 Nanolipoprotein particles (NLPs) have arisen as a platform to readily solubilize membrane proteins while mimicking a native lipid environment. NLPs consist of a discoidal phospholipid bilayer encircled by an apolipoprotein belt. In an effort to optimize and …

Crystallization During Volume Reduction Of Solutions With An Ion-Composition Corresponding To That In The Distal Tubuli, Inge Hojgaard, Anne-Marie Fornander, Mari-Anne Nilsson, Hans Goran Tiselius

Scanning Microscopy

The effect of macromolecules on the crystallization in solutions with an ion-composition and a pH corresponding to that of urine in the distal part of the distal tubuli was examined by recording the number and volume of crystals in a Coulter Multisizer and by studying the crystal morphology with scanning electron microscopy at different degrees of evaporation. The experiments were carried out with 100 ml samples of salt solutions with and without different concentrations of dialysed urine (dU) from normal subjects. Addition of dU resulted in a greater number of crystals and a reduction in the mean crystal volume (MCV). …

The Nucleation And Growth Of Calcium Phosphate Crystals At Protein And Phosphatidylserine Liposome Surfaces, G. H. Nancollas, A. Tsortos, A. Zieba

Scanning Microscopy

The kinetics of calcium phosphate crystal growth at the surfaces of proteins and phospholipids has been investigated using free drift and constant composition methods in supersaturated calcium phosphate solutions (relative supersaturations: with respect to hydroxyapatite, HAP, σHAP = 15.0, and with respect to octacalcium phosphate, OCP, σOCP = 1. 9). Fibrinogen and collagen molecules adsorbed at hydrophobic surfaces as well as uncross-linked collagen fibrils induce ion binding and subsequent nucleation of calcium phosphate. The formation of OCP on phosphatidylserine vesicles introduced to highly supersaturated calcium phosphate solutions probably involves the interaction of the calcium ions with the ionized carboxylic groups …

Formation Of Hydrated Calcium Oxalates In The Presence Of Poly-L-Aspartic Acid, Jeffrey A. Wesson, Elaine Worcester

Scanning Microscopy

The effect of poly-L-aspartic acid (PA) on the crystal structure of calcium oxalate crystals grown after spontaneous nucleation was evaluated as a function of relative supersaturation and calcium:oxalate ratio in a buffered salt solution, with pH and ionic strength in the range of normal human urine. PA was used as a model for naturally occurring acidic urine proteins that have been shown to inhibit nucleation and growth of calcium oxalate crystals. The crystals grown were characterized by optical microscopy and X-ray powder diffraction. It was observed that calcium oxalate monohydrate was the preferred crystalline form in the absence of added …

Crystallization And Stone Formation Inside The Nephron, Dirk J. Kok

Scanning Microscopy

A model is presented visualizing the events leading to calcium-salt, crystal- and stone-formation inside the nephron. For each nephron segment, handling of urine components relevant to stone formation is considered and urine composition determined. This information was applied to nucleation experiments simulating passage of urine through a nephron. The model and in vitro experiments suggest that within normal transit times for the respective nephron segments, particles of a hydroxyapatite-like material first form near the bend in the Loop of Henle of juxtamedullary nephrons. From there on, calcium oxalate particles start to appear: first dihydrate, then monohydrate. In the collecting duct …

Zeta Potential Measurement And Particle Size Analysis For A Better Understanding Of Urinary Inhibitors Of Calcium Oxalate Crystallization, L. C. Cao, G. Deng, E. R. Boeve, W. C. De Bruijn, R. De Water, C. F. Verkoelen, J. C. Romijn, F. H. Schroder

Scanning Microscopy

To better understand urinary inhibitors of calcium oxalate crystallization, both zeta potential measurement and particle size analysis were chosen to illustrate: (1) the potential therapeutic efficacy of G872, a semi-synthetic sulfated polysaccharide, in stone prevention; and (2) the relative contribution of various urinary fractions {e.g., ultrafiltered urine (UFU), Tamm-Horsfall protein (THP), urinary polyanionsprecipitated with cetylpyridinium chloride (CPC), urinary macromolecular substances with different concentration ratios (UMSl0,50,90 and UMS'l0,50,90) and THP-free urine (THPFU)} to total urinary inhibitory activity. The results showed: (1) addition of G872 significantly enhances urinary inhibitory activity and negative zeta potential values; (2) re-addition of the CPC to UFU …

Induction Of Crystallization Of Calcium Oxalate Dihydrate In Micellar Solutions Of Anionic Surfactants, H. Füredi-Milhofer, L. Tunik, N. Filipovic-Vincekovic, D. Skrtic, V. Babic-Ivancic, N. Garti

Scanning Microscopy

Calcium oxalate dihydrate (CaC₂O₄.(2+x)H₂O; COD; x ≤ 0.5) does not readily crystallize from electrolytic solutions but appears as a component in crystalluria. In this paper, we review in vitro studies on the factors responsible for its nucleation and growth with special attention given to the role of surfactants. The following surfactants were tested: dodecyl ammonium chloride (cationic), octaethylene monohexadecylether (non-ionic), sodium dodecyl sulfate (SOS, anionic), dioctyl sulphosuccinate (AOT, anionic), and sodium cholate (NaC, anionic). The cationic and some of the anionic surfactants (SOS, AOT) induced different habit modifications of growing calcium oxalate crystals by …

Experimental Calcium Oxalate Nephrolithiasis And The Formation Of Human Urinary Stones, Saeed R. Khan

Scanning Microscopy

Calcium oxalate nephrolithiasis in rats requires induction of hyperoxaluria which results in increased urinary calcium oxalate supersaturation. As a result of low to mild chronic hyperoxaluria, calcium oxalate crystals deposit first in the papillary collecting ducts. Crystal deposition in the kidneys is preceded by calcium oxalate crystalluria and starts with the retention of aggregated calcium oxalate crystals in the renal tubules. Retained crystals move from the tubules to the interstitium, and in the process, become anchored to the tubular basement membrane. Crystal aggregates present in the superficial peripheral collecting ducts of the renal papillae ulcerate through to the papillary surface …

Effect Of Urinary Macromolecules And Chondroitin Sulphate On Calcium Oxalate Crystallization In Urine, A. L. Rodgers, D. Ball, W. Harper

Scanning Microscopy

After filtration and ultrafiltration (10 kD) of 24 hour urine specimens from 12 healthy male subjects, calcium oxalate crystallization was induced in the filtered (FILTD) and ultrafiltered (UF) fractions by administration of a sodium oxalate load. In addition crystallization was also induced in UF fractions to which physiological quantities of chondroitin sulphate (CHON) had been added (UF + CHON). The rate of calcium oxalate crystallization was determined by measuring the rate at which turbidity increased. Crystal numbers and sizes were measured with a Malvern particle size analyzer and by scanning electron microscopy.

Crystallization rates, crystal numbers and crystal sizes were …

Crystallization Of Calcium Oxalate And Calcium Phosphate At Supersaturation Levels Corresponding To Those In Different Parts Of The Nephron, Ján Lupták, Hanne Bek-Jensen, Anne-Marie Fornander, Inge Höjgaard, Mari-Anne Nilsson, Hans-Göran Tiselius

Scanning Microscopy

The risk of crystallization in solutions, with a composition corresponding to that of urine in various parts of the nephron, was assessed by studying urine samples containing standardized increments of calcium and oxalate concentrations. The experiments were carried out in salt solutions with and without dialysed urine and the appearance of crystals was detected and measured with a Coulter counter. With increased concentrations of calcium, crystals of calcium phosphate were predominantly formed in solutions with a composition corresponding to that in the proximal and distal tubuli, whereas, calcium oxalate was the primary crystallization product in solutions with a composition corresponding …

Importance Of Mild Hyperoxaluria In The Pathogenesis Of Urolithiasis - New Evidence From Studies In The Arabian Peninsula, W. G. Robertson, H. Hughes

Scanning Microscopy

The hypothesis that mild hyperoxaluria is more important than hypercalciuria in the pathogenesis of urolithiasis is re-examined in the light of new evidence. Small increments in urinary oxalate in the normal to high-normal range are much more critical than similar rises in urinary calcium for increasing the relative supersaturation of urine with respect to calcium oxalate, the oxalate/calcium ratio in urine, the total volume of calcium oxalate crystals excreted, the proportion of abnormally large crystals and aggregates of calcium oxalate and the severity of the disorder as defined by the recurrence rate of stone-formation. Data from the Arabian Peninsula, where …