Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction.

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

Beyond Saffman-Delbruck approximation: a new regime for 2D diffusion of α-hemolysin complexes in supported lipid bilayer.

Cell mechanisms are actively modulated by membrane dynamics. We studied the dynamics of a first-stage biomimetic system by Fluorescence Recovery After Patterned Photobleaching. Using this simple biomimetic system, constituted by α -hemolysin from Staphylococcus aureus inserted as single heptameric pore or complexes of pores in a glass-supported DMPC bilayer, we observed true diffusion behavior, with no immobile fraction.

Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

Background: Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step.

Exploring bovine pancreatic trypsin inhibitor phase transitions.

This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools.

A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin.

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 A resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.

Identification and characterization of a triacylglycerol lipase in Arabidopsis homologous to mammalian acid lipases.

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms. By contrast, very little is known on plant TAG lipases. An Arabidopsis cDNA called AtLip1 (At2g15230), which exhibits strong homology to lysosomal acid lipase, was found to drive the synthesis of an active TAG lipase when expressed in the baculovirus system.

Oil-bodies as substrates for lipolytic enzymes.

Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown.

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings.

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form.

Study of fatty acid specificity of sunflower phospholipase D using detergent/phospholipid micelles.

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates.

Characterization and kinetic properties of a soya-bean cell-wall phosphatase.

A phosphatase from soya-bean cell walls was purified to homogeneity and characterized. It consists of two identical 70-kDa subunits linked by one or several disulphide bridges and, to our knowledge, it does not seem to require metal ions to be fully active. At high substrate concentrations, the enzyme was most efficient at slightly alkaline pH levels, which is at variance with the acid requirements of phosphatases previously established in other plant cell walls; whereas at low substrate concentrations it was more active at acid pH levels.

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase.

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange.

Isolation and purification of chloroplastic spinach (Spinacia oleracea) sedoheptulose-1,7-bisphosphatase.

Higher-plant sedoheptulose-1,7-bisphosphatase was isolated and purified over 200-fold from spinach (Spinacia oleracea) chloroplast stromal extracts to apparent electrophoretic homogeneity by DEAE-Fractogel, molecular sieving on Sephadex G-200 and Blue B dye-matrix affinity chromatography. It is a protein of Mr 66,000, made up of two apparently identical subunits (Mr 35,000). The enzyme is activated by reduced thioredoxin fb in the presence of dithiothreitol.

Structural, immunological and kinetic comparisons of NADP-dependent malate dehydrogenases from spinach (C3) and corn (C4) chloroplasts.

This paper compares structural, immunological and kinetic properties of corn (C4) and spinach (C3) NADP-malate dehydrogenases. These chloroplastic enzymes are regulated in vivo by thiol-disulfide interchange. Both in their oxidized (inactive) and reduced (active) states these enzymes have a dimeric structure with molecular masses for the subunit ranging from 28 kDa to 38 kDa according to the procedure used for the determination.