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PubMed1988

Calcium pyrophosphate crystal deposition: the effect of monosodium urate and apatite crystals in a kinetic study using a gelatin matrix model.

The kinetics of calcium pyrophosphate dihydrate (CPPD) crystal growth was studied by allowing calcium and pyrophosphate (PPi-4) ions to diffuse through a denatured collagen matrix (biological grade gelatin) in the presence of either monosodium urate monohydrate (MSU) or hydroxyapatite (HA) crystals. In this in vitro model system, MSU crystals significantly altered the kinetics of PPi-4 ionic diffusion through the gelatin matrix by allowing the [PPi-4] gradient to fall off much more rapidly, suggesting an increased level of scavenging of PPi-4 ions into crystalline materials. Even more significantly, the presence of MSU crystals markedly influenced the crystal growth morphology of triclinic CPPD, producing that observed in vivo. A large number of epitaxially dimensional matches between MSU and triclinic (t) and monoclinic (m) CPPD were identified, suggesting that MSU crystals can epitaxially induce CPPD crystal growth. This finding supports the hypothesis that the association of urate gout and CPPD crystal deposition disease is based on the nucleating potential of MSU crystals for CPPD crystal growth. In contrast, the HA crystal structure did not appear to serve as a nucleating agent for CPPD crystals. However, HA crystals did serve as effective traps for PPi-4 ions and their presence led to more stable CPPD crystal growth.

Apatites↗
PubMed1988

Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals. Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor.

Tissue deposits of basic calcium phosphate (BCP) crystals are associated with various clinical manifestations of inflammation. We addressed the possibility that native proteins modify the ability of hydroxyapatite (HA) crystals to stimulate human inflammatory cells. Neutrophil superoxide release and chemiluminescence in response to HA crystals (0.3-4.0 mg/ml) were blunted by serum and plasma. Inhibitory activity was progressively removed from serum by sequential adsorption with HA crystals, suggesting that the inhibitors were crystal-bound proteins. Thus, we characterized HA crystal-bound plasma proteins by O'Farrell gels: Fibronectin, transferrin, albumin, alpha 2-HS glycoprotein (AHSG), alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, Gc globulin, haptoglobin, and high density lipoprotein apolipoproteins were major bound species. Of these, AHSG was the most active inhibitor of HA-induced neutrophil superoxide release, and this glycoprotein partially (60%) restored inhibitory activity to HA-adsorbed serum. AHSG also bound in vitro to the related BCP crystal, octacalcium phosphate, but only minimally to calcium pyrophosphate dihydrate crystals and monosodium urate crystals. Suppressive effects on neutrophil stimulation exhibited by AHSG were also specific for BCP crystals. AHSG was present in noninflammatory synovial fluids bound to synthetic HA crystals in vitro, and AHSG could be detected on native synovial fluid HA crystals. We conclude that the binding of AHSG may modulate the inflammatory potential of BCP crystals.

Blood Physiological Phenomena↗
PubMed2001

Phosphorylated osteopontin peptides suppress crystallization by inhibiting the growth of calcium oxalate crystals.

BACKGROUND: Osteopontin isolated from human urine [uropontin (uOPN)] is a potent inhibitor of calcium oxalate (CaOx) monohydrate (COM) crystallization. However, specific structural features responsible for its effects on CaOx crystallization were not previously known. The present studies were designed to define molecular features responsible for interactions of uOPN with COM crystals and the inhibition of crystallization. METHODS: Peptides and phosphopeptides with sequences corresponding to potential crystal binding domains within the protein sequence of osteopontin were synthesized. Then the effects of these peptides on COM crystal growth and crystal aggregation were investigated and their secondary structures analyzed. RESULTS: Growth of COM crystals was inhibited by approximately 50% at 1000 nmol/L concentrations by the two unmodified peptides with the closest clustering of aspartic acid residues. Growth was not inhibited by the other two unmodified peptides, with aspartic residues more evenly distributed within their sequences. Phosphorylation markedly increased inhibition of COM crystal growth, so that each of the four phosphorylated peptides inhibited growth by at least 50% at concentrations of < or =200 nmol/L. Phosphorylation of these peptides did not cause changes in secondary structure that would favor interaction with COM crystal surfaces. CONCLUSIONS: These studies of synthetic peptides identify molecular features within the osteopontin molecule that contribute to the inhibition of one aspect of COM crystallization. The inhibition of crystal growth induced by phosphorylation appears to result from altered local patterns of charge density, since conformational changes favoring interaction with crystals were not caused by phosphorylation.

Calcium Oxalate↗
PubMed2001

Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI.

Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. It could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGDI, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGDI, NDelta23 and NDelta66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. In total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type NDelta23 and NDelta66 RhoGDI which did not crystallize. Four crystal structures were solved (the triple mutants NDelta23:K135,138,141A and NDelta66:K135,138,141A, and two single mutants NDelta66:K113A and NDelta66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize.

Alanine↗
PubMed2005

Structure of a high-resolution crystal form of human triosephosphate isomerase: improvement of crystals using the gel-tube method.

Crystals of human triosephosphate isomerase with two crystal morphologies were obtained using the normal vapour-diffusion technique with identical crystallization conditions. One had a disordered plate shape and the crystals were hollow (crystal form 1). As a result, this form was very fragile, diffracted to 2.8 A resolution and had similar crystallographic parameters to those of the structure 1hti in the Protein Data Bank. The other had a fine needle shape (crystal form 2) and was formed more abundantly than crystal form 1, but was unsuitable for structure analysis. Since the normal vapour-diffusion method could not control the crystal morphology, gel-tube methods, both on earth and under microgravity, were applied for crystallization in order to control and improve the crystal quality. Whereas crystal form 1 was only slightly improved using this method, crystal form 2 was greatly improved and diffracted to 2.2 A resolution. Crystal form 2 contained a homodimer in the asymmetric unit, which was biologically essential. Its overall structure was similar to that of 1hti except for the flexible loop, which was located at the active centre Lys13.

Crystallization↗
PubMed2002

The effect of seed crystals of hydroxyapatite and brushite on the crystallization of calcium oxalate in undiluted human urine in vitro: implications for urinary stone pathogenesis.

BACKGROUND: The aim of this study was to determine whether crystals of hydroxyapatite (HA) or brushite (BR) formed in urine promote the epitaxial deposition of calcium oxalate (CaOx) from undiluted human urine in vitro and thereby explain the occurrence of phosphate in the core of urinary stones consisting predominantly of CaOx. MATERIALS AND METHODS: Crystals of HA, BR, and CaOx were generated from human urine and their identity confirmed by X-ray analysis. Standard quantities of each crystal were then added to separate aliquots of pooled undiluted human urine and CaOx crystallization was induced by the addition of identical loads of sodium oxalate. Crystallization was monitored by Coulter Counter and (14) C-oxalate analysis and the precipitated crystals were examined by scanning electron microscopy. RESULTS: In comparison with the control to which no seeds were added, addition of CaOx crystals increased the deposition of (14) C-oxalate by 23%. On the other hand, seeds of HA and BR had no effect. These findings were supported by Coulter Counter analysis, which showed that the average modal sizes of crystal particles precipitated in the presence of HA and BR seeds were indistinguishable from those in the control, whereas those deposited in the presence of CaOx were significantly larger. Scanning electron microscopy confirmed these results, demonstrating that large aggregates of CaOx dihydrates were formed in the presence of CaOx seeds, whereas BR and to a lesser extent HA seeds were scattered free on the filtration membrane and attached like barnacles on the surface of the freshly precipitated CaOx crystals. CONCLUSION: Seed crystals of HA or BR do not promote CaOx deposition in urine in vitro and are therefore unlikely to influence CaOx crystal formation under physiologic conditions. However, binding of HA and BR crystals to, and their subsequent enclosure within, actively growing CaOx crystals might occur in vivo, thereby explaining the occurrence of mixed oxalate/phosphate stones.

Calcium Oxalate↗
PubMedDate not supplied

Mechanisms of cellular interaction with monosodium urate crystals. IgG-dependent and IgG-independent platelet stimulation by urate crystals.

Monosodium urate crystals (MSU) stimulate suspensions of washed platelets or neutrophils. When MSU crystals are coated with IgG, as occurs in plasma, stimulation is markedly enhanced. These studies which use MSU-induced human platelet serotonin secretion as a model examine the nature of cellular recognition mechanisms for MSU crystals and IgG-coated MSU crystals. F(ab')2 fragments of specific anti-Fc antibody blocked and the lipopolysaccharide of Salmonella minnesota R595 enhanced human platelet secretion induced by IgG-coated urate crystals. These agents had little effect on stimulation by uncoated crystals. This indicated that urate crystals stimulate platelets independently of fluid phase IgG. Urate crystals directly stimulated suspensions of washed rabbit platelets which lack Fc receptors. In contrast to human cells, stimulation was blocked by IgG. This again demonstrated IgG-independent cell stimulation by urate crystals. Calcium pyrophosphate dihyrate crystals could trigger human platelet secretion only when coated with IgG. This suggests that when crystals are coated with IgG, the surface-bound IgG alone may be the stimulus to the cell. This was supported by the finding that polyvinylpyridine-N-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated, but not IgG-coated, urate crystals. These data indicate that urate crystals (and potentially other surface or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of adsorbed IgG or, when coated with IgG, by triggering the cell via immunoglobulin receptors.

Blood Platelets↗
PubMedDate not supplied

Crystallization behavior and thermal properties of melt-crystallized poly[(R)-3-hydroxybutyric acid-co-6-hydroxyhexanoic acid] films.

Melt-crystallized films of poly[(R)-3-hydroxybutyric acid-co-10mol% 6-hydroxyhexanoic acid] (P[(R)-3HB-co-6HH]) were prepared by isothermal crystallization at various temperatures for 3 days, and subsequently stored at room temperature after the films formed well-developed and volume-filled spherulites. The lamellar morphologies and properties of melt-crystallized films were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, transmission electron microscopy, and atomic force microscopy. The melting endotherm of P[(R)-3HB-co-6HH] films was composed of a broad peak starting around room temperature and of a sharper peak starting above the isothermal crystallization temperature. The stacking of flat-on lamellae with lamellar periodicity of 8-10 nm was detected on the surface of P[(R)-3HB-co6HH] films after the primary crystallization at 110 degrees C. On storage at room temperature above the Tg (-5 degrees C) of copolyester, thin crystals of 1-4 nm thickness appeared on the surface of P[(R)-3HB-co-6HH] films crystallized at 110 degrees C. These results suggest that long sequences of (R)-3HB units in a random copolyester form relatively thick P[(R)-3HB] crystalline lamellae during the primary crystallization process at a given crystallization temperature, while shorter sequences of (R)-3HB units, which are incapable of crystallizing at a given crystallization temperature, form relatively thin crystalline lamellae during the subsequent crystallization process at room temperature.

Calorimetry, Differential Scanning↗
PubMed2003

Cell-surface matrix proteins and sialic acids in cell-crystal adhesion; the effect of crystal binding on the viability of human CAKI-1 renal epithelial cells.

OBJECTIVE: To investigate the role of sialic acids and cellular matrix proteins as crystal-binding molecules in human calcium-oxalate nephrolithiasis. MATERIALS AND METHODS: The well-defined human renal cancer cell line CAKI-1 was used a standard cell culture system. After enzymatic digestion of various cell surface molecules, the binding of alpha2,6 (Sambucus nigra, SN-) and alpha2,3 (Maackia amurensis, MA)-specific lectins to CAKI-1 cells was analysed. Simultaneously, the effect on adhesion and release of calcium oxalate monohydrate crystals was investigated (eight replicates). The effect of crystal adhesion on cell viability was assessed using Trypan blue exclusion (five replicates). RESULTS: Neuraminidase decreased MA-lectin binding of CAKI-1 cells by 39% (P < 0.05) but elevated SN-lectin binding by 812% (P < 0.05). Simultaneously, crystal binding to CAKI-1 cells was increased by 28% (P > 0.05). Pretreatment with collagenase type I, trypsin and dispase II reduced crystal-binding by 61-74% (P < 0.05) with no effect on sialic acid-specific lectin-binding. However, only collagenase type I and dispase (ratio 4 : 1) were also able to release crystals from their receptor-binding sites (P < 0.05). An increase in the number of cell surface-bound crystals correlated significantly with a decrease in cell viability (P < 0.05). CONCLUSIONS: alpha2,3-linked sialic acids protect cells from crystal-binding. Much greater SN-lectin binding associated with only moderately increased crystal binding argues against alpha2,6-linked sialic acids as a main target structure of crystals. In contrast, collagen type I, type IV and/or fibronectin seem to be potent crystal-binding molecules on human renal epithelial cells, with collagen type I involved in a potential second step of crystal-cell interaction.

Cell Survival↗
PubMed2006

A crystal structure study when crystallized only in d(CG)3 + methylamine and a d(CG)3 + metal ion.

There is not an example of the crystallization only of d(CG)(3) and a metallic cation without putting the amine in addition in there is no example of the crystallization d(CG)(3) with methylamine that is the mono-amine so far either. We were able to obtain a very beautiful crystal with all sides of 0.3mm by the sitting drop method that had done the crystallization of d(CG)(3)+ methylamine and d(CG)(3)+ Mg(2 +). The crystal was diffracted up to 1.0 A by using the synchrotron radiation. As a result of the X-ray crystal structure analysis, methylamine was not seen while d(CG)(3)+ methylamine was crystallizing mysteriously. d(CG)(3) had stabilized with five Mg(2+) ions. d(CG)(3) had stabilized similarly with five Mg(2+) ions in the crystal of d(CG)(3)+ Mg(2+). The position of these five Mg(2+) ions understood that I agreed on superimpose in a crystal of d(CG)(3) + methylamine, a crystal of d(CG)(3)+Mg(2+), a crystal of d(CG)(3) + spermidine, and crystals of d(CG)(3) + PA (24) which was already analyzed so as to cut it and stabilized d(CG)(3) at an about the same position.

Cations↗
PubMed2000

A rational approach towards successful crystallization and crystal treatment of human cytomegalovirus protease and its inhibitor complex.

The crystallization and subsequent crystal treatment of both free human cytomegalovirus (hCMV) protease and its inhibitor complexes are reported. For free-enzyme crystals, diffraction was greatly improved by optimizing the crystallization conditions, raising the precipitant concentration in the reservoir and soaking the crystals in artificial mother liquors. Each of the six components in the final crystallization formula (16% PEG 4000, 0.1 M MES pH 6.0, 0.4 M LiCl, 10% glycerol, 5% t-butanol and 5 mM Na(2)S(2)O(3)) plays a distinctive role and is indispensable. A synergistic effect of Na(2)SO(4) and t--butanol on diffraction was observed and studied. A 2.0 A multiwavelength anomalous diffraction (MAD) data set was collected using a synchrotron-radiation source, leading to the elucidation of the three-dimensional structure of the enzyme. For the inhibitor-complex crystals, initial attempts with co-crystallization and soaking experiments at pH 6.0 did not produce conclusive results. Subsequently, experiments were designed to co-crystallize the complex at pH 8.0, the optimal pH for the enzyme and the inhibitor activity. Using 20-50 mM spermine in the crystallization buffer, crystals of two peptidomimetic inhibitor complexes were obtained at pH 7.5 and 8.0. Spermine was required for the inhibitor complexes to be crystallized at pH 8.0, possibly neutralizing net negative charges of hCMV protease owing to its acidic pI of 5.5. A 2.7 A data set was collected from one of the inhibitor complexes and the structure was determined using the molecular-replacement method.

Crystallization↗
PubMedDate not supplied

Zinc protoporphyrin IX binds heme crystals to inhibit the process of crystallization in Plasmodium falciparum.

The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 microM, which is a ratio of 1:40,000 hemes, can elevate 10-fold in some of the anemias associated with malaria disease protection. This work examines a binding mechanism for zinc protoporphyrin IX inhibition of heme crystallization similar to the antimalarial quinolines. Zinc protoporphyrin IX neither forms crystals alone nor extends on preformed heme crystals. Inhibition of both seed heme crystal formation and crystal extension occurs with an inhibitory concentration (IC)50 of 5 microM. Field emission in-lens scanning electron microscopy depicts the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of preformed hemozoin templates. In vitro zinc protoporphyrin IX, like the quinolines, binds to heme crystals in a saturable, specific, pH, and time-dependent manner. The ratio at saturation is approximately 1 zinc protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc protoporphyrin IX to heme ratio 6 to 10 times that in the erythrocyte cytosol, which also corresponds to elevated ratios found in heme crystals purified from Plasmodium parasites. This work implicates protection from malaria by a mechanism where elevated zinc protoporphyrin IX in anemic erythrocytes binds to heme crystals to inhibit further crystallization. In endemic malaria areas, severe iron deficiency anemia should be treated with antimalarials along with iron replenishment.

Animals↗
PubMed2004

Modification of surface crystal formation on mother crystal by additive molecules.

Additive molecules and excess reactant species (NaOH and NH4F) in NaF crystallization were used to control surface crystal formation on a mother crystal. When the surface integration was inhibited or bulk diffusion facilitated, more surface crystals were found on the mother crystal due to an increase in the interfacial supersaturation on the crystal. The inclusion of KCl as an additive and increases in the reactant concentration and agitation speed resulted in a higher population of surface crystals on the mother crystal, because surface integration was inhibited. However, surface crystal formation was suppressed by the polymer gelatin due to retardation of the bulk diffusion by the gelatin layer formed around the crystal. In addition, surface crystal formation significantly changed according to the feeding mode, and the excess species of NaOH originating from the NH4F feeding mode were more effective in facilitating surface crystal formation than the excess species of NH4F coming from the NaOH feeding mode due to the smaller ionic radii of NaOH than of NH4F.

Journal Article↗
PubMed2004

Growth dynamics of isotactic polypropylene single crystals during isothermal crystallization from a miscible polymeric solvent.

The present article presents a spatiotemporal growth of isotactic polypropylene (iPP) single crystals, melt crystallized from a polymeric solvent, i.e., poly (ethylene octene) copolymer that is known to be miscible with iPP. Optical and atomic force microscopic investigations reveal that the melt grown single crystals of iPP develop in the form of two parallel rows of crystal lamellae, but these crystals merge at the tips. To elucidate the mechanism of these emerging parallel rows of iPP crystals, a phase field model pertaining to solidification phenomena has been employed that involves a nonconserved crystal order parameter and a chain-tilting angle. This phase field model is based on the free energy of crystallization, having an asymmetric double well, and a tensorial surface free energy of the crystal interface coupled with a curvature elastic free energy that is possessed by the solid-liquid interface. The spatiotemporal simulation of iPP single crystal growth has been carried out on a square lattice based on the finite difference method for spatial steps and an explicit method for temporal steps with a periodic boundary condition. The appearance of the seemingly twin crystal is captured in the simulation, which may be attributed to the sector demarcation that is taking place in the anisotropically growing single crystal of iPP.

Journal Article↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of new crystal forms of Escherichia coli P(II) complexed with various ligands.

New crystals of the signal-transducing protein P(II) have been obtained in the presence of a number of different effector ligands. Various crystal forms are observed depending on the nature of the ligand(s). Co-crystallization with 2-ketoglutarate, glutamate and pyrophosphate produces hexagonal crystals similar to the wild type, ATP yields cubic crystals and ATP in conjunction with 2-ketoglutarate or glutamate yields orthorhombic crystal forms. All of the above crystals have been characterized by X-ray diffraction analysis. The hexagonal crystals belong to space group P6(3), cubic crystals to either I23 or I2(1)3 and orthorhombic crystals to I222. A molecular-replacement solution for the P(II)/ATP/2-ketoglutarate crystals has been obtained giving us an initial model for a trimer in the orthorhombic crystal form.

Journal Article↗
PubMed2004

Crystal engineering of photochromic diarylethene single crystals.

Photochromic performance of diarylethene single crystals was controlled by crystal engineering using non-covalent aromatic-aromatic interactions as the directional intermolecular force. A diarylethene derivative with two pentafluorophenyl groups, 1,2-bis(2-methyl-5-pentafluorophenyl-3-thienyl)perfluorocyclopentene (1a), formed stoichiometric co-crystals with benzene (Bz) and naphthalene (Np) by aryl-perfluoroaryl interactions. Face-to-face pi-stacking interactions between the pentafluorophenyl groups of 1a and the aromatic molecules are responsible for 2:1 and 1:1 stoichiometric compositions in 1a/Bz and 1a/Np co-crystals, respectively. The diarylethene underwent thermally stable and photoreversible photochromic reactions in a homo-crystal of 1a and co-crystals 1a/Bz and 1a/Np. The absorption spectra of the photogenerated closed-ring isomers varied depending on the conformation of the diarylethene molecules packed in the crystals. The diarylethene 1a also formed 1:1 stoichiometric co-crystals with different kinds of diarylethenes, 1,2-bis(2-ethyl-5-phenyl-3-thienyl)perfluorocyclopentene (2a) and 1,2-bis[2-methyl-5-(1-naphthyl)-3-thienyl]perfluorocyclopentene (3a). Both co-crystals 1a/2a and 1a/3a showed photochromism. Although 1a, 2a, and 3a underwent efficient photocyclization reactions in their homo-crystals, highly selective photocyclization reactions of 2a or 3a were observed in the co-crystals. The selective reactions were confirmed by HPLC and X-ray crystallography. Excited energy transfers from 1a to 2a and from 1a to 3a are considered to occur and cause the selective reactions.

Crystallization↗
PubMed1994

Quantitative analysis of crystal growth. Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis.

We show that quantitative analysis of replicated, full-factorial crystal growth experiments and, by implication, similar studies of a wide variety of other phenomena, can be a powerful tool for analyzing macromolecular systems with complex, interacting dependencies on functionally significant factors. Bacillus stearothermophilus tryptophanyl-tRNA synthetase crystallizes in three different crystal forms depending on the ligands present under otherwise identical conditions. Comparison of crystallographic space groups for complexes with different ligands reveals that the three forms entail at least two very different families of packing arrangements that are correlated with specific changes in the enzyme ligation state. One is associated with the ligand-free enzyme, substrate ligands, and the binding of the activated amino acid; the other results from the presence of high ATP concentrations and/or the synthesis of the unusual acyl-transfer product, tryptophanyl-2'(3') ATP. Together with previous physico-chemical studies of aminoacyl-tRNA synthetases, these observations suggest that the two families are related, respectively, to the biochemical processes of amino acid activation and acyl transfer. Further evidence that the crystal polymorphism results from an underlying protein conformational polymorphism has now been obtained by quantitative analysis of how crystal growth depends on pH and the substrates tryptophan and ATP. The analysis consists first in showing that crystallization conditions for the unliganded protein are very favorable, suggesting that variation in crystal growth induced by pH and substrates under otherwise identical conditions is due to their effects on the protein conformation and not on incidental perturbations of crystal growth, per se. Next, crystal growth experiments are shown to be reproducible enough to support statistical analysis of quantitative scores assigned to the results. Finally, the observed variation in scores can be attributed at high confidence levels chiefly to three effects: that of pH alone, the synergistic effects of pH plus tryptophan, and of tryptophan plus ATP. These statistical inferences are consistent with other biochemical data, and support the conclusions based on crystal packing that representative stages of the enzyme mechanism have been trapped in the different crystal forms. The pH-tryptophan interaction implies that there is a pH-dependent conformational change favoring high affinity substrate binding at high pH. The pH-ATP interaction implies that a subsequent conformational change, not previously considered, occurs between tryptophan activation and acyl transfer.

Adenosine Triphosphate↗
PubMedDate not supplied

Human erythrocyte catalase: 2-D crystal nucleation and production of multiple crystal forms.

Negatively stained electron microscope images are presented, showing the nucleation of two-dimensional (2-D) crystals of human erythrocyte catalase produced on mica by the negative staining-carbon film technique. Examples of the formation of partially ordered 2-D arrays and more ordered 2-D crystals are shown and the conditions required for the production of large well-ordered 2-D crystals discussed. The structural transformation of one flexuous 2-D paracrystal into a p2 2-D crystal is considered. The crystallographic 2-D image average of this p2 crystal form is presented (lattice parameters a = 9.0 nm, b = 18.6 nm, gamma = 90.8 degrees). It is shown that transmission electron microscopy provides the possibility of defining 2-D crystal nucleation, growth of intermediate forms, and low-resolution crystallographic structural analysis of 2-D crystals of human erythrocyte catalase. Comparison of the various electron microscopical negatively stained images with the peptide backbone of the X-ray structure of bovine liver catalase at different tilt and rotation positions correlates with and emphasizes the multiple intermolecular contacts and orientations that can be adopted by human erythrocyte catalase, leading to various 2-D arrays and 2-D crystals. Alignment of the surface groups involved in the protein-protein interactions that occur during 2-D crystal nucleation and crystal growth may ultimately be determined. From this approach, when taken together with detailed consideration of protein-solvent and protein-solute electrostatic interactions in solution, and at the fluid-air interface, it is considered that a more general theory of crystal nucleation and growth may eventually emerge.

Aluminum Silicates↗