Immunochromatographic sticks for tissue transglutaminase and antigliadin antibody screening in celiac disease.

Background & Aims: We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-transglutaminase (t-TG) as an alternative to enzyme-linked immunosorbent assays (ELISAs) for celiac disease (CD) screening.

Methods: We used a tissue-transglutaminase (t-TG) stick, which detected immunoglobulin A/G (IgA/G) antibodies to t-TG, and a t-TG/antigliadin antibodies (AGA) stick, which detected IgA antibodies to both t-TG and AGA, as well as t-TG and AGA ELISAs, to determine t-TG and AGA antibodies in untreated celiac patients with subtotal villous atrophy. A total of 142 children (3 IgA-deficient sera) and 30 adults, and 140 controls (normal mucosa; 121 children and 19 adults), plus 23 sera from pediatric CD patients in remission were assayed.

Apoflavodoxin folding mechanism: an alpha/beta protein with an essentially off-pathway intermediate.

The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization.

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family.

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method.

Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase.

The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd.

Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity.

Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain. Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M.

Charge reversal mutations in a conserved acidic patch in Anabaena ferredoxin can attenuate or enhance electron transfer to ferredoxin:NADP+ reductase by altering protein/protein orientation within the intermediate complex.

A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabaena ferredoxin (Fd), comprising residues D67, D68, and D69, have been constructed by site-directed mutagenesis. One such mutant, D68K, has a rate constant for electron transfer (et) to Anabaena ferredoxin:NADP+ reductase (FNR) at low ionic strength (I = 12 mM) which is 2.5 times larger than wild type (9000 vs 3600 s-1).

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded.

Site-specific mutagenesis demonstrates that the structural requirements for efficient electron transfer in Anabaena ferredoxin and flavodoxin are highly dependent on the reaction partner: kinetic studies with photosystem I, ferredoxin:NADP+ reductase, and cytochrome c.

Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-flow spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been shown to result in very large inhibitions of electron transfer to FNR, were found to yield wild-type behavior in reactions with PSI and cytc. In general, the effects of Fd mutagenesis on the PSI reactions were considerably smaller than those observed for the FNR reaction.