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R J Truscott

Publications and source records attributed to R J Truscott.

At least 55 records · Page 3Linked to original sources
PubMed1994

A 1H NMR spectroscopic comparison of gamma S- and gamma B-crystallins.

Two-dimensional 1H NMR spectroscopic studies are presented on bovine gamma S- and gamma B-crystallin. In gamma S-crystallin, the four N-terminal residues have great flexibility compared with the rest of the molecule and assume a random coil conformation. NMR spectroscopy and electrospray mass spectrometry show that the N-terminal residue is acetylated. Thus, gamma S-crystallin is similar to the acidic beta-crystallins in having a flexible N-terminal extension and an N-terminus that is blocked with an acetyl group but no C-terminal extension. In addition to the short N-terminal extension in gamma S-crystallin, other unassigned resonances are also observed in the NMR spectra. In gamma B-crystallin, however, cross-peaks in the NH to alpha-CH region of the spectrum are essentially restricted to the last three residues of the C-terminal domain. The NMR data imply that gamma S-crystallin has a more flexible structure than gamma B-crystallin. Sedimentation equilibrium studies on gamma S-crystallin are consistent with this proposal. Resonances from the N-terminal extension of gamma S-crystallin are not affected by the presence of alpha-crystallin implying that this region is not involved in interactions between the two molecules. It is concluded that gamma S-crystallin shares structural properties which are intermediate between the beta- and gamma-crystallins.

Animals↗
PubMed1994

Supramolecular order within the lens: 1H NMR spectroscopic evidence for specific crystallin-crystallin interactions.

alpha-, beta- and gamma-crystallins from bovine lens contain flexible terminal extensions which are readily observed by NMR spectroscopy. By monitoring these resonances, NMR spectroscopy therefore offers a means of examining specific protein-protein interactions in crystallin mixtures. In this paper, a 1H NMR spectroscopic study of bovine lens nuclear and cortical homogenates and various crystallin mixtures is presented. In both homogenates, resonances from the flexible C-terminal extensions of alpha-crystallin and the N-terminal extension of beta B2-crystallin are readily observed suggesting that these regions are not involved in crystallin-crystallin interactions. In the cortical homogenate, resonances from the short N-terminal extension of gamma S-crystallin are also present. The cortical homogenate gives rise to more intense resonances than the nuclear homogenate, suggesting that the cortical region has many more mobile crystallin regions. In both homogenates, the C-terminal extension of beta B2-crystallin and the very short C-terminal extension of gamma B-crystallin are not observed. Thus, the C-terminal regions of these proteins are involved in interactions with other crystallins. Similar effects are observed upon mixing of the individual crystallins, e.g. the C-terminal extension of gamma B-crystallin is absent in spectra of mixtures of total gamma-crystallin and high-molecular-weight beta-crystallin aggregates (beta H). Overall, the results are consistent with a short-range order for the crystallins within the lens.

Animals↗
PubMed1994

Ultraviolet filter compounds in human lenses: 3-hydroxykynurenine glucoside formation.

Experiments have been conducted on various aspects of the biosynthetic pathway leading to the formation of 3-hydroxykynurenine glucoside (3OHKG), the major tryptophan-derived human lens UV filter compound. Measurable levels of the reactive metabolite, 3-hydroxykynurenine (3OHKyn) were found in normal human lenses, including lens nuclei. 3OHKyn was able to enter organ-cultured lenses and to serve as a substrate for production of 3OHKG. Thus lenticular 3OKHyn can potentially be derived either by metabolism of Trp in the lens or from transport into the lens. Lens incubation followed by dissection showed that the equatorial region was probably the major site of synthesis of 3OHKG. The concentration of 3OHKG was relatively constant along the visual axis but was significantly lower in the equatorial region: a pattern also found for Kyn and 3OHKyn. No evidence, however could be obtained for hydrolysis of 3OHKG by lens homogenates.

Adolescent↗
PubMed1994

Gourami lenses convert tryptophan into 3-hydroxykynurenine.

Although free tryptophan was found in the lens of both the kissing gourami (Helostoma temmincki) and the 3-spot gourami (Trichogaster trichopterus), only the lens of the latter species contained 3-hydroxykynurenine. Radiolabel experiments carried out on 3-spot gourami lenses in culture demonstrated that, as in humans, lenticular tryptophan is converted to 3-hydroxykynurenine probably via the intermediate kynurenine.

Animals↗
PubMed1993

An investigation into the stability of alpha-crystallin by NMR spectroscopy; evidence for a two-domain structure.

The stability of bovine lens alpha-crystallin with respect to temperature, pH and urea has been investigated by 1H and 31P-NMR spectroscopy. The 1H and 31P-NMR spectra of alpha-crystallin show little change with temperature up to 75 degrees C, indicating that alpha-crystallin has great thermal stability and does not undergo any major change in structure with temperature. 1H spectral studies of alpha-crystallin and its isolated alpha A and alpha B subunits reveal a marked difference in the stability of these species. It is found that, at pH 2.5, alpha A-crystallin adopts a native conformation whereas alpha B-crystallin is denatured. On the other hand, the two subunits when part of the total alpha-crystallin aggregate adopt a native conformation at pH 2.5, but in the presence of 0.1 M glycine the alpha B subunits become denatured. Thus, alpha A-crystallin and total alpha-crystallin are more stable species than alpha B-crystallin and, in total alpha-crystallin, there is an interaction between the compact domains of the alpha A and alpha B subunits that leads to enhanced stability. Finally, changes in the 1H and 31P-NMR spectra of alpha A-crystallin and alpha B-crystallin in the presence of varying concentrations of urea are consistent with a two-domain model for alpha-crystallin subunits with the C-terminal domain being less stable and unfolding first in the presence of urea.

Animals↗
PubMed1993

1H-NMR spectroscopy of beta B2-crystallin from bovine eye lens. Conformation of the N- and C-terminal extensions.

1H-NMR spectroscopic studies of a 46-kDa homodimer, beta B2-crystallin, from bovine eye lens are presented. beta B2-crystallin has terminal extensions extending from globular N- and C-terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two-dimensional NMR methods on beta B2-crystallin, its synthesised terminal extensions and a proteolysed sample of beta B2-crystallin with a portion of its C-terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One-dimensional experiments at various pH values provided H-2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of beta B2-crystallin are in excellent agreement with those for the X-ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776-780]. No change in the spectrum of beta B2-crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.

Animals↗
PubMed1993

1H-NMR spectroscopy of bovine lens beta-crystallin. The role of the beta B2-crystallin C-terminal extension in aggregation.

1H-NMR spectroscopic studies of bovine eye lens beta-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three beta-crystallin aggregates are dominated by resonances from the beta B2 subunit, particularly from the N- and C-terminal extensions of this subunit. Resonances from other beta subunits, which all have terminal extensions, are, in general, absent from spectra of the beta-crystallin aggregates. Therefore, the beta B2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other beta-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of beta B2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of beta B2 and the hydrophobic beta B1 subunit, which is only found in the octomer, is discussed. At higher temperatures (45 degrees C) in the octomer, partial exposure of the C-terminal extension of beta B2 occurs indicating that the octomer may be starting to break up into smaller aggregates.

Animals↗
PubMed1993

UV filters in human lenses: tryptophan catabolism.

Primate lenses are unique in that they convert tryptophan (trp) into 3-hydroxykynurenine glucoside (30HKG). This is the major short-wave absorbing pigment present in human lenses and it may play a role in protecting the eye from UV-induced photodamage. A study has been performed on aspects of this metabolic pathway in human lenses. A significant rate of synthesis could be observed in a 24-hr period using intact lenses to which radiolabelled tryptophan had been added. Label was found in kynurenine (Kyn), 3-hydroxykynurenine (30HKyn) and 30HKG, although always to the greatest extent in the latter metabolite. Considerable variation in the proportion of label incorporated into 30HKG was observed. Older lenses tended to accumulate a greater percentage into the glucoside; the data indicating a generally greater flux through the trp catabolic pathway in lenses above 60 years of age. Pulse-chase experiments on lens pairs suggested that there may be a significant loss or metabolism of 30HKG. Biosynthesis of 30HKG was found to take place in the lens epithelial cells. A linear rate of 30HKG efflux from organ cultured lenses was observed indicating that one pathway for removal of this compound involves diffusion through the lens capsule. That this pathway also occurs in vivo was confirmed by analysing samples of human vitreous humour. Based on efflux rates from cultured lenses (1.07 x 10(-3) +/- 0.293 x 10(-3) mumol hr-1, n = 5), half-life values for 30HKG in the lens ranging between 7 and 40 hr were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

The modification of proteins by 3-hydroxykynurenine.

The autoxidation and reactivity towards proteins of 3-hydroxykynurenine, a tryptophan metabolite found in the human lens, has been studied. At neutral pH, 3-hydroxykynurenine was readily oxidized using molecular oxygen with the formation of several coloured products. The autoxidation of both 3-hydroxykynurenine and the related aminophenol, 3-hydroxyanthranilic acid, was inhibited by the inclusion of sulphydryl compounds such as glutathione or cysteine. Covalent adducts involving the thiols were not observed with either aminophenol. 3-Hydroxykynurenine was found to react with proteins, including lens proteins, to produce brown-coloured polypeptides characterized by an indistinct long wavelength absorption. This protein tanning was inhibited by glutathione. Despite the presence of an amino group in the side chain of 3-hydroxykynurenine, this tanning of proteins was found to involve amino groups including those of lysine residues, as has been found for 3-hydroxyanthranilic acid. Although both aminophenols tanned polylysine, only 3-hydroxykynurenine induced precipitation of the polyamino acid. 3-Hydroxykynurenine tanned all of the purified crystallins but induced precipitation only in the case of alpha A-crystallin. The implications of these findings for senile cataract are discussed.

3-Hydroxyanthranilic Acid↗
PubMed1993

Spectral transmission and short-wave absorbing pigments in the fish lens--I. Phylogenetic distribution and identity.

Fish lens transmission was found to vary depending on the type and concentration of short-wave absorbing compounds present within the lens. Pigments extracted from lenses of ten species were identified as mycosporine-like amino acids (mainly palythine, palythene and asterina-330, lambda maxs around 320-360 nm) which are also thought to be present in the majority of the 120 species examined here. A novel mycosporine-like pigment with lambda max 385 nm was isolated from the lens of the flying fish, Exocoetus obtusirostris, while lenses of several closely related tropical freshwater species were found to have high concentrations of the tryptophan catabolite 3-hydroxykynurenine (lambda max 370 nm). The type of lens pigment a species possesses and its concentration depends upon both the animal's phylogenetic group and its "optical niche".

Animals↗
PubMed1992

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.

Amino Acid Sequence↗
PubMed1992

Involvement of tyrosine residues in the tanning of proteins by 3-hydroxyanthranilic acid.

The binding of oxidized phenolic compounds to proteins is of importance in a number of biological systems, including the sclerotization of insect cuticle and the tanning of cocoons. 3-Hydroxyanthranilic acid (3HAA), an aminophenol, is a tryptophan metabolite that undergoes autoxidation readily, and proteins incubated in the presence of 3HAA and oxygen become colored and oxidized. Some moth species are thought to employ this reactivity of 3HAA with proteins for the tanning of cocoons, but the detailed mechanism of this process has not been studied previously. We show that one reaction pathway involves the covalent coupling of 3HAA with tyrosine to form a benzocoumarin derivative, a dibenzo[b,d]pyran-6-one. The stability of the benzocoumarin to conditions of acid hydrolysis normally used for protein digestion has enabled the isolation of the tyrosine adduct from bovine serum albumin that had been incubated with 3HAA. The adduct was also isolated from cocoons of Samia cynthia and Hyalophora gloveri, two species of moths reported to utilize 3HAA for cocoon tanning. These findings indicate that one mechanism of interaction of 3HAA with proteins involves a radical-radical coupling with tyrosine residues.

Journal Article↗
PubMed1992

Kynurenine identified as the short-wave absorbing lens pigment in the deep-sea fish Stylephorus chordatus.

A number of deep-sea fish have bright yellow lenses whose coloration is attributable to a variety of largely unidentified short-wave absorbing pigments. Here the pigment of the deep-sea fish Stylephorus chordatus has been isolated and identified by NMR and mass spectroscopy as kynurenine; a pigment also found in the human lens. The degree of this pigmentation is greater in older animals. The fact that the lenses of both a deep-sea fish and man contain the same pigment is of interest, given the vastly different light environments they inhabit.

Animals↗
PubMed1992

Direct approach to identification, at the molecular level, of modified proteins in human nuclear cataractous lenses: beta-crystallin is a component of the urea-insoluble protein fraction.

In an effort to elucidate the molecular changes which take place in the human lens with the onset of nuclear cataract, the urea-insoluble protein fraction, solubilized with dithiothreitol, was digested with trypsin. Tryptic peptides separated by HPLC, were examined by both mass spectrometry and Edman degradation. A pentapeptide Gly-Glu-Tyr-Pro-Arg which is contained within the beta-crystallin sequence was isolated. This finding provides direct evidence that beta-crystallin is present in the urea-insoluble protein fraction which is known to be characteristic of human nuclear cataract lenses.

Amino Acid Sequence↗