[The effect of the phenolic compounds exuded by pea roots in darkness on the reproduction of Rhizobium].

The exudation, composition, and biological activity of the phenolic compounds (PC) of pea (Pisum sativum L.) roots in the light and darkness were studied. The roots of leguminous plants grown for 5 days in darkness exuded a smaller amount of PC that displayed a weaker stimulation of Rhizobium reproduction. Moreover, the root exudates contained antimicrobial compounds, stilbenes. It is assumed that a lower PC exudation by roots and the specific features of PC composition influencing the biological activity are among the reasons causing a delayed nodulation of legumes grown in darkness.

[Active oxygen species in pea seedlings during the interactions with symbiotic and pathogenic microorganisms].

The level of active oxygen species (AOS)--superoxide anion radical (O2*-) and hydrogen peroxide (H2O2)--in pea (Pisum sativum L.) cultivar Marat seedlings was studied upon their inoculation with symbiotic (Rhizobium leguminosarum bv. viceae strain CIAM 1026) and pathogenic (Pseudomonas syringae pv. pisi Sackett) microorganisms. Different patterns of the changes in AOS in pea seedlings during the interactions with the symbiont and the phytopathogen were recorded. It is assumed that O2*- and H2O2 are involved in the defense and regulatory mechanisms of the host plant.

The DHHC domain: a new highly conserved cysteine-rich motif.

A unique clone from a human pancreatic cDNA library was isolated and sequenced. Examination of the deduced polypeptide sequence of the clone showed a new form of cysteine-rich domain that included a region with the form of a Cys4 zinc-finger-like metal binding site followed by a complex Cys-His region. Searches of the Swiss-Protein data bank found a similar 48-residue domain in fifteen open reading frames deduced from A. thaliana, C. elegans, S. cerevisiae and S. pombe genomic sequences. The high degree of conservation of this domain (13 absolutely conserved and 17 highly conserved positions) suggests that it has an important function in the cell, possibly related to protein-protein or protein-DNA interactions. The gene recognized by the clone is is localized to human chromosome 16, and is conserved in vertebrates. The 2 Kb message is expressed in various human fetal and adult tissues. An antibody made to a peptide sequence of the deduced protein showed reactivity in immunoblots of monkey lung and retinal subcellular fractions and immunohistochemically in late fetal mouse tissues and a limited number of adult mouse tissues, including pancreatic islets, Leydig cells of the testis, and the plexiform layers of the retina.

Analysis of a human cDNA containing a tissue-specific alternatively spliced LIM domain.

A unique clone, isolated from a human pancreatic cDNA library, was sequenced and characterized. Northern blot analysis showed that the gene is active in a number of fetal and adult tissues, and immunoblots showed expression in nuclear and cytosolic cell fractions. The gene corresponding to the clone was localized to chromosome 13 by human/rodent somatic cell hybrid panels. The largest open reading frame contains a LIM domain, and the deduced peptide from the open reading frame appears to have the characteristics of a LIM-only protein, designated LMO7. RT-PCR and genomic sequence analyses indicate that expression of this gene product is subject to tissue-specific modulation by elimination of the LIM domain by alternative splicing in neural tissues.

Probing the inhibitor-binding site of aldose reductase with site-directed mutagenesis.

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Study of a fatty acid binding site of interphotoreceptor retinoid-binding protein using fluorescent fatty acids.

Interphotoreceptor retinoid-binding protein (IRBP) is a 140-kDa glycolipoprotein which constitutes about 70% of the soluble protein of the retinal interphotoreceptor matrix. Much is known concerning its role in the transport of retinoids between photoreceptor cells and pigment epithelium but little is known about its interactions with lipids. Here we have examined the physicochemical characteristics of a fatty acid binding site of IRBP using a set of fluorescent fatty acid analogs with an anthracene moiety attached at different positions along the hydrocarbon chain. The results show that fatty acids are bound in a hydrophobic environment as indicated by a blue shift in fluorescence maxima and by a increase in quantum yield of the bound ligand. There is a single specific fatty acid binding site for each molecule of IRBP with an apparent Kd = 3.6 x 10(-7) M. There is a nonradiative energy transfer from tryptophan residues to bound ligand. The interactions of IRBP and bound fatty acid are sensitive to denaturation by increasing concentrations of urea as judged by changes in nonradiative energy transfer efficiency and the quantum yield of bound probe. Quantum yields of bound fatty acid analogs varied with position of the fluorophore along the hydrocarbon chain and had the lowest values for the fluorophore located at the midpoint. Probing of the microenvironment of bound fluorophore with a quencher indicated a highly structured binding site.

Studies on the location of aromatic amino acids in alpha-crystallin.

The locations of tryptophan residues in alpha-crystallin and homopolymers constructed from the alpha A- and alpha B-chains were examined by comparing their fluorescence emission properties and their accessibilities to quenchers. Two classes of tryptophan could be distinguished on the basis of differences in their spectral characteristics, fluorescence decay lifetimes, quenching with acrylamide and exposure by increasing concentrations of urea. Polarization measurements indicated that the tryptophan residues were associated with flexible segments of the polypeptide chains. The two classes could be assigned, one to Trp-9 (in both A- and B-chains) which is in an hydrophobic environment, and one to Trp-60 (B-chain) which appeared to be nearer the surface of the aggregate. No evidence was found for residues inaccessible to the quenchers. An apparent partition coefficient of 40 was obtained for the association of acrylamide with the protein. From temperature-dependence studies, it was concluded that there was a significant energy barrier to the penetration of acrylamide into the protein matrix (Ea = 5.8 kcal/mol) and that entry of the quencher was through channels produced by temporary disruption of the matrix (delta s = 1.5 eu). The phenolic side-chains of tyrosine residues in several different alpha-crystallins were found to ionize with pK values above pH 11, indicative of residues highly shielded from the solvent. Comparisons of polypeptide sequences, together with tyrosine fluorescence emission data and the pK values, permitted a tentative assignment of residue locations. All of the data are consistent with a possible micelle-like structure for alpha-crystallin but not with a layered structure.

The effects of sonication on alpha-crystallin.

Sonication of bovine alpha-crystallin increases its molecular mass from around 770 kDa to in excess of 2,300 kDa. Exposure to 2M urea or 0.1 M glycine pH 7, did not affect the size of the sonicated protein, indicating that it did not consist of dimers and higher polymers of the original molecule. Sonication of a mixture of alpha-crystallins labelled on the A chain sulphydryl group with either an aminonaphthalene or a fluorescein chromophore, generated a product exhibiting substantial energy transfer. The average distance between the probes was calculated to be 5 nm. These observations suggest that sonication has generated a new quaternary structure, incorporating subunits from two or more different alpha-crystallin molecules. No significant differences were observed in the microenvironments of tryptophan residues although those in the sonicated protein could be more easily exposed by controlled denaturation with urea. A small decrease was observed in the quenchability of a fluorescent probe attached to the sulphydryl group and a small increase in the uptake of an hydrophobic probe. These data suggest that sonication may have altered the conformation of the subunits at, or near the surface of the protein.

Excimer fluorescence in lens proteins labelled with N-(1-pyrene)maleimide.

Lens proteins labelled with the sulphydryl reagent, N-(1-pyrene)maleimide exhibit pyrene excimer fluorescence around 470 nm. This was found to be associated with both the beta- and gamma-crystallins but not the alpha-crystallins. Disappearance of the 470 nm peak on exposure to denaturants indicates it arose from an intramolecular excimer. This excimer fluorescence provides a new and sensitive tool for monitoring conformational changes in the beta- and gamma-crystallins.

Specific dissociation of alpha B subunits from alpha-crystallin.

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.

Quenching of tryptophan fluorescence in bovine lens proteins by acrylamide and iodide.

The microenvironments of tryptophan residues in bovine alpha-, beta H-, beta L and gamma-crystallins have been examined using acrylamide and KI quenching of fluorescence. From a consideration of the differential effects of the two quenchers, the quenching efficiencies and spectral changes, it was possible to distinguish tryptophans in different environments and to assign these to specific residues in the sequence. Two classes of tryptophan were identified in gamma-crystallin, one buried and one moderately accessible. The buried class contained tryptophans 42A and 125 which lie in the angles of the wedge-shaped domains of the protein. These residues, which had emission maxima at 326 nm, were not accessible to quenching by iodide. The more accessible residues, emitting at 334 nm, corresponded to tryptophans 64 and 148 which are in the widest part of the wedge-shaped subunit and close to the surface of the protein. The two beta-crystallins were virtually indistinguishable. They contained two buried tryptophans, probably residues 58 and 150, and three close to the surface, residues 81, 84 and 166. The quenching efficiencies for these two classes were lower than those observed with gamma-crystallin. Since the three-dimensional structures of the beta- and gamma-crystallins are probably very similar, this suggests that the polymeric nature of the beta-crystallins is responsible for the decreased accessibility of the tryptophans to the quenchers. alpha-crystallin demonstrated unusually high static quenching which made it difficult to distinguish different classes of tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)