Search PubMed⌕ Search

Biomedical subjects

J J Harding

Publications and source records attributed to J J Harding.

At least 37 records · Page 2Linked to original sources
PubMed1997

Effect of aging on the chaperone-like function of human alpha-crystallin assessed by three methods.

alpha-Crystallin can function as a molecular chaperone by preventing unwanted interactions. This paper presents the effects of aging and cataract on the chaperone-like properties of alpha-crystallin from soluble fractions from the cortex and nucleus of human lenses by using three assays: enzyme inactivation and two turbidity experiments. The three methods complemented each other. There was no decrease with age of chaperone-like function of cortical alpha-low and alpha-high crystallin. Nuclear alpha-low crystallin showed a decrease, whereas alpha-high crystallin showed no age-related change but its protective effect was diminished. Results from the nucleus of 40-year-old cataractous lenses seemed similar to those for clear lenses of equivalent age, whereas 80-year-old cataractous lenses showed decreased chaperone-like behaviour.

Adult↗
PubMed1997

A spectroscopic study of glycated bovine alpha-crystallin: investigation of flexibility of the C-terminal extension, chaperone activity and evidence for diglycation.

The effect of glycating the C-terminal extensions of alpha-crystallin on their flexibility was investigated. In the course of the study the reaction sites were identified and double glycation of single lysine residues was found. Alpha-crystallin was incubated until approximately one mole of the sugar had reacted per subunit of the crystallin. The reaction sites were investigated by mass spectrometry and H NMR spectroscopy, and were found to be principally in the short and flexible C-terminal extensions. The chaperone ability of alpha-crystallin was unaffected by this limited glycation. There was little effect on the flexibility of the C-terminal extensions. This result supports the view that the flexibility of the C-terminal extensions of alpha-crystallin is important for chaperone activity. As alpha-crystallin consists of a mixture of unmodified and phosphorylated subunits, a detailed investigation was undertaken of the reaction of galactose with peptides comprising the C-terminal extensions of alphaA- and alphaB-crystallin. The alphaA peptide was incubated with galactose until 0.79 mole of sugar was bound per mole of peptide and the alphaB peptide reacted until 2.2 moles of galactose had been incorporated. The purified glycated peptides were examined by NMR and mass spectrometry to identify glycation site(s), and the effect of glycation on the conformation of the peptides. For both peptides, it was found that extensive glycation of the constituent lysine residues occurred. The addition of two galactose molecules to some lysine residues of the peptides was also noted. This diglycation was confirmed in control experiments with N-acetyl-lysine.

Animals↗
PubMed1997

Glycation-induced inactivation and loss of antigenicity of catalase and superoxide dismutase.

Oxidative mechanisms are thought to have a major role in several biological phenomena, including cataract formation and diabetic complications. Here we investigate the inactivation of catalase and superoxide dismutase, both powerful antioxidant enzymes, by sugars of different glycating abilities, and the loss of antigenicity that was monitored by the loss of activity after immunoprecipitation with monospecific antibodies. The antigenicity of non-glycated or glycated enzymes separated by affinity chromatography were determined by dot-blotting. Incubation with sugars resulted in a time-dependent inactivation of the enzymes. Ribose and fructose inactivated them more rapidly than glucose and glucose 6-phosphate. Glycation induced losses of antigenicity and inactivation simultaneously. The glycated enzymes had entirely lost their antigenicity compared with non-glycated enzyme. These results further support the idea that inactivation of enzyme and loss of antigenicity are simultaneous. This might occur in the pathogenesis of diabetic complications and aging.

Aging↗
PubMed1997

The effects of ageing on the chaperone-like function of rabbit alpha-crystallin, comparing three methods of assay.

The lens has a high protein content necessary for focusing light on to the retina. Alpha-Crystallin accounts for approximately 40% of the protein and has been shown to act in a chaperone-like manner. Here we show the effects of ageing on the chaperone-like properties of alpha-crystallin from rabbit lens. Three assays were used to determine chaperone ability. Non-enzymatic glycosylation inactivation of malate dehydrogenase is protected by alpha-crystallin. Thermal aggregation of beta-low crystallin and malate dehydrogenase are both prevented by alpha-crystallin. Three ages of rabbit lens were used. Alpha-Crystallin from the soluble fraction of the cortex and nucleus were investigated as well as alpha-high and alpha-low fractions resolved by size-exclusion chromatography. All three methods complemented each other. There was no age-dependent loss in chaperone-like behaviour for both alpha fractions in the cortex. There was an early decrease with age of the nuclear alpha-low fraction. Nuclear alpha-high shows no age-related decrease but its chaperoning ability is greatly compromised. Post-translational modifications which occur during ageing may be responsible for the effect of alpha-crystallin chaperone-like ability in the lens nucleus.

Aging↗
PubMed1997

Molecular chaperones protect catalase against thermal stress.

Lenticular alpha-crystallin is generally thought of as having limited chaperone functions. It can efficiently suppress the aggregation of proteins but is unable to promote the functional refolding of proteins after denaturation in many systems unlike other molecular chaperones. However, it has been reported that alpha-crystallin, along with the small heat-shock proteins, is able to promote the functional refolding of some enzymes after thermal and chemical denaturation. These chaperones are also able to confer protection against the thermal inactivation of these enzymes. In results presented here, we demonstrate that alpha-crystallin, along with chaperonin 60 (GroEL), was able to provide statistically significant and specific protection against catalase thermal inactivation at stoichiometrical concentrations. The small heat-shock protein, heat-shock protein 25 (Hsp25), was unable to confer any such protection. alpha-Crystallin however was unable to promote the functional refolding of thermally inactivated catalase. alpha-Crystallin and Hsp25 both efficiently suppressed the thermal aggregation of catalase. A high-molecular-mass (HMM) complex was only observed to develop in solutions containing catalase and alpha-crystallin after solutions were 80-fold more concentrated relative to thermal inactivation assay conditions prior to incubation. SDS/PAGE analysis confirmed that alpha-crystallin had formed a soluble complex with catalase after a period of thermal stress.

Animals↗
PubMed1997

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).

Amino Acid Sequence↗
PubMed1997

Bovine and human alpha-crystallins as molecular chaperones: prevention of the inactivation of glutathione reductase by fructation.

With no measurable protein synthesis occurring in the centre of the lens, structural proteins and enzymes there will need to be stable for many years, if not decades, in order to maintain lens integrity and function. Recent work has indicated that alpha-crystallin, which is sequentially related to heat shock proteins, has chaperone-like properties in that it is capable of preventing heat-induced aggregation of various proteins, including other crystallins. Thus this universal vertebrate lens protein may contribute to maintenance of lens integrity by protecting other lens proteins from non-enzymic insults or the consequences thereof. We previously showed that the enzyme glutathione reductase was inactivated in a time-dependent manner when incubated with various sugars, suggesting glycation was responsible for this effect. In this paper we confirmed that this was the case. Using this enzyme model system, the inclusion of either bovine or human alpha-crystallin protected against the inactivation of glutathione reductase by fructation. This action was specific, with control proteins displaying no such protection. Use of high performance liquid chromatography supported the fact that alpha-crystallin did not act simply by mopping up free sugar but rather maintained the activity of the modified enzyme. Dose-dependent experiments indicated that human alpha-crystallin was more effective than its bovine counterpart, which might be expected considering the much longer lifespan of humans. The stoichiometry of the protection by both alpha-crystallins indicated that alpha-crystallin with glutathione reductase was not acting like GroEL as a large complex with a hydrophobic pore, but rather that individual subunits may be capable of acting as chaperones.

Animals↗
PubMed1997

Recent studies of risk factors and protective factors for cataract.

The multifactorial nature of cataractogenesis has been further exposed in recent epidemiologic studies, most efficiently by four case-control studies. The factors found include various plasma constituents, steroids, alcohol, diabetes, hypertension, hyperlipidemia, and obesity. Further examination of dietary factors leaves confusion as to their role in the prevention of cataract.

Cataract↗
PubMed1996

Inhibition of 6-phosphogluconate dehydrogenase by carbamylation and protection by alpha-crystallin, a chaperone-like protein.

Carbamylation of lens proteins may contribute to cataract formation in populations with high levels of blood urea. Urea comes to equilibrium with cyanate. Changes induced by cyanate binding to lens crystallin have been described but little is known about the carbamylation of the enzymes. The present study investigated the in vitro carbamylation of 6-phospho-D-gluconate dehydrogenase (E.C.1.1.1.44) and its effect on the enzymic activity, as well as a possible way to prevent the cyanate binding to the enzyme. The covalent cyanate binding to protein inactivated the enzyme in a concentration-dependent fashion. Aspirin and paracetamol did not protect the enzyme against inactivation by carbamylation, while alpha-crystallin was specifically protective as compared with other control proteins, consistent with its suggested role as a molecular chaperone.

Acetaminophen↗
PubMed1996

Glycation-induced inactivation of malate dehydrogenase protection by aspirin and a lens molecular chaperone, alpha-crystallin.

Non-enzymic glycosylation (glycation) of structural proteins has been widely studied as a possible mechanism in the long-term complications of diabetes. Here we show that glycation inactivates malate dehydrogenase. Aspirin affords some protection against the glycation, but alpha-crystallin, a lens protein which appears to act as a molecular chaperone in other systems, is much more effective. For example, 5 mM glucose completely inactivates malate dehydrogenase in four days, and 5 micrograms alpha-crystallin/ml provides complete protection against this inactivation. Fructose, a superior glycating agent, inactivates the enzyme in 24 hours but even so the same low concentration of alpha-crystallin is able to protect 80% of the activity. Other proteins provide no protection at the same concentration. The inactivation of malate dehydrogenase and other enzymes by glycation could play a role in diabetic complications, and molecular chaperones like alpha-crystallin could serve to protect them.

Animals↗
PubMed1996

Alpha-crystallin acting as a molecular chaperone protects catalase against steroid-induced inactivation.

A link between corticosteroid therapy and the development of cataract has been known for many years. However, the precise underlying molecular mechanism of pathology has not been characterised, although a role for direct deleterious interactions between corticosteroids and lenticular proteins has been investigated. Alpha-crystallin is a major lens protein that has exhibited chaperone properties in vitro. Catalase is a ubiquitous enzyme that is an important scavenger of hydrogen peroxide in vivo. The corticosteroid prednisolone-21-hemisuccinate was found to inactivate bovine liver catalase, in vitro in a progressive manner. Coincubation of alpha-crystallin with catalase in a 1:2 molar ratio (one alpha-crystallin to two catalase molecules) fully protected against this inactivation. The protection was specific. Aspirin-like analgesics, putative anti-cataract drugs offered no such protection.

Animals↗
PubMed1996

The penetration of topical cysteamine into the human eye.

The penetration of cysteamine into human aqueous was measured to assess its suitability as a topical agent for the prevention of radiation-induced cataract. Cysteamine drops (0.5% and 1.0%) were administered preoperatively to cataract patients every half hour, for two, four, six or eight hours. Aqueous humor was collected at the start of surgery and assayed for total free thiol. There was no significant difference in the aqueous thiol level of any of the treatment groups compared with the controls. No side effects were observed.

Administration, Topical↗
PubMed1996

Testing time for the sunlight hypothesis of cataract.

The proceedings of a 2-day workshop on the role of sunlight in cataract formation have provided an opportunity to assess the importance of this widely discussed risk factor. There seems to be no role in nuclear cataract, and any role in cortical or posterior subcapsular cataract is scarcely measurable.

Animals↗
PubMed1996

Prevention of the fructation-induced inactivation of glutathione reductase by bovine alpha-crystallin acting as a molecular chaperone.

We had previously shown that glutathione reductase was inactivated by glycation, and decided to use this model system to see whether bovine alpha-crystallin could provide protection, and whether its modification had an effect. Other proteins were used as a comparison. Both forms of alpha-crystallin prevented loss of enzyme activity on incubation with fructose. No such protection was profferred by the other proteins, suggesting bovine alpha-crystallin acted in a chaperone-like manner.

Animals↗
PubMed1996

The influence of some post-translational modifications on the chaperone-like activity of alpha-crystallin.

We investigated the influence of phosphorylation, glycation, carbamylation and oxidative modification on the capacity of alpha-crystallin to protect beta-crystallins against heat denaturation. Simple modification of lysine residues by early glycation or carbamylation had no effect. However, late (cross-linking) glycation products and oxidative modifications decreased the chaperone-like activity of alpha-crystallin. Homopolymers of alpha A-crystallin had a higher protecting capacity compared with those of alpha B-crystallin. The in vivo phosphorylated forms of especially alpha A- but also alpha B-crystallin revealed a somewhat better protecting ability than the respective non-phosphorylated forms.

Animals↗