Biomedical subjects
Publications and source records attributed to R J Truscott.
An unusual pink compound has been isolated from the proteolytic digests of senile nuclear cataract proteins by adsorption onto Sephadex G-10. The tiny amounts obtained precluded definitive structural elucidation, however TLC comparisons show that it resembles triphenodioxazine-1,8-dicarboxylic acid, a pink heterocyclic chemical formed by oxidative condensation of 3-hydroxyanthranilic acid.
The mechanism of reaction of proteins with 3-hydroxyanthranilic acid (3OHA) under oxidizing conditions has been examined. A range of proteins were found to tan when exposed to oxidized 3OHA. One exception was lysozyme which tanned only after being denatured by reduction and carboxymethylation. Chemical modification experiments using bovine serum albumin (BSA) suggested that lysine was the primary site of reaction in 3OHA-mediated protein tanning. This reactivity of 3OHA toward lysine was confirmed by autoxidizing 3OHA in the presence of amino acid homopolymers. The rate of modification of both BSA and polylysine was pH dependent. At neutral pH, a component of the coloration of the protein was found to be due to the formation of a lysyl-p-quinone adduct. Other products appear to arise through addition to the 3OHA quinone imine. Poly-(Glu,Lys) was tanned by 3OHA at a greatly reduced rate, suggesting that electrostatic interactions may influence the reaction with lysine residues and may provide an explanation for the lack of tanning of lysozyme. Despite the reaction between 3OHA and lysine, amino acid analysis revealed little quantitative change in the lysine content of proteins even after exposure to 3OHA for a period of 24 h. These results support the proposal that reaction with lysine residues is the major route of protein tanning by 3-hydroxyanthranilic acid.
One of the research programs of the Australian Cataract Research Foundation (ACRF) is aimed at investigating the possibility that senile cataract both cortical and nuclear, may result from the interaction of reactive metabolites with proteins in the lens. In particular we are exploring the potential role of tryptophan metabolites for example, 3-hydroxyanthranilic acid, in this disease. This article examines briefly some aspects of this approach.
The autoxidation of the tryptophan metabolite, 3-hydroxyanthranilic acid, at pH 7 gives rise to a p-quinone dimer and cinnabarinic acid. A novel dimer formed by radical-radical coupling of 3-hydroxyanthranilic acid is also produced. Labelling studies have shown that the C-2 oxygen in the p-quinone dimer is derived from molecular oxygen. A product versus time study of this reaction has revealed that, in the absence of catalase, cinnabarinic acid is formed but undergoes decomposition by hydrogen peroxide. At pH 7, in the presence of catalase, both the p-quinone dimer and cinnabarinic acid are formed at approximately the same rate and this rate of formation increases with increasing pH. Inclusion of superoxide dismutase was found to increase the rate of formation of cinnabarinic acid, suggesting that superoxide ions may also cause decomposition of cinnabarinic acid. This was confirmed by treating cinnabarinic acid with superoxide. A mechanism involving a common anthranilyl radical intermediate is proposed to account for the formation of the different oxidation products.
Human cataract lens proteins can be bleached by exposure to sodium borohydride (NaBH4), sodium cyanoborohydride (NaCNBH3), or hydrogen peroxide (H2O2). The decolouration resulting from these treatments could be monitored by a change in absorbance at 350 nm. At pH 12 the magnitude of the absorbance change increased in proportion with the severity of the nuclear cataract in the case of NaBH4 and H2O2 treatments, but not in the case of NaCNBH3 treatment. The rate of change in absorbance at 350 nm following exposure to the different reagents was used to evaluate three model systems for senile nuclear cataract. These model systems utilized calf lens proteins which had been tanned by exposure either to 3-hydroxyanthranilic acid, dopa/tyrosinase, or ultraviolet light.
When intact rat lenses were incubated in artificial aqueous humor in the presence of 1 mM calcium and a sulfhydryl reagent p-chloromercuriphenyl sulfonate (pCMPS) a visible annular opacity developed within 4 hours. Combined photographic and ion-sensitive microelectrode investigations of the lenses demonstrated that the subsequent linear increase in opacification was accompanied by an increase in internal free calcium. Opacities were not observed in lenses incubated in the absence of either pCMPS or calcium. Gel electrophoresis of the soluble and urea-soluble fractions from lenses exposed to 1 mM calcium for periods of up to 14 hours showed no evidence for crystallin degradation and only minor proteolysis of cytoskeletal proteins. When lenses were incubated under identical conditions, but with 5 mM calcium, the degree of opacification increased up to approximately 8 hours and then remained constant. A progressive loss in cytoskeletal proteins was observed which correlated with a further increase in free calcium such that by 14 hours of incubation, when the internal calcium approached 1 mM, most of the spectrin and vimentin present in the cortex of the lens had disappeared. An unidentified 110-kilodalton protein also disappeared from lenses incubated in 5 mM calcium. These results indicate that proteolysis by calcium-dependent enzymes such as calpain may play a significant role in cytoskeletal regulation and metabolism in the lens. A role for cytoskeleton/membrane/crystallin interaction in calcium-induced opacification is discussed.
An isocratic method for the separation of most of the tryptophan metabolites in the oxidative degradation pathway is described. The chromatographic analysis utilizes the combined selectivity and sensitivity of amperometric and fluorimetric detection. The effect of pH, ionic strength and operating potential on retention times and detector responses are evaluated. The use of dual electrochemical cells at two operating potentials together with a programmable fluorescence detector allows for improved selectivity for the detection of metabolites. The sensitivity achieved with this method allows for the detection of the metabolites in biological fluids at the picomole level. The method has been used to monitor serum samples obtained during a tryptophan load test.
Incubation of intact rat lenses under conditions that stimulated a net influx of calcium resulted in a pronounced loss of transparency and a major decrease in the levels of spectrin. The progressive loss of this cytoskeletal component coincided with the appearance of polypeptides of approximately 150 kDa which showed immunoreactivity with an antibody raised to spectrin. These bands disappeared on further incubation. It is, therefore, suggested that a calcium-activated protease is present in the lens which is capable of degrading spectrin by the initial removal of approximately 90 kDa fragments. This process calcium-induced proteolysis may be the basis for the cytoskeletal reorganisation observed during the differentiation of lens fibre cells and may be involved in cataract development.
Proteins, including lens proteins, were incubated in the presence of 3-hydroxyanthranilic acid (30 HA) under oxidizing conditions. Samples were monitored for alterations in color, fluorescence, sulfhydryl content, lysine availability, methionine content, tryptophan content and protein size. Incubation of proteins with 30 HA produced rapid brown coloration and a correspondingly rapid decrease in sulfhydryl content. Alpha-, beta- and gamma-crystallins were all found to react with 30 HA. An increase in protein fluorescence (excitation 340/emission 425 nm) accompanied the color development. No significant decrease in the content of tryptophan or any other amino acid was detected by amino acid analysis. The levels of available lysine were not affected significantly by treatment with 30 HA. Oxidation of methionine to methionine sulfoxide and the covalent cross-linking of polypeptides was obtained by subsequent treatment of the tanned proteins with H2O2. The modifications observed are very similar to those found in the senile nuclear cataract lens.
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The reagent 2, 4, 6-trinitrobenzenesulfonic acid was used to probe the status of protein amino groups in the nuclei of control (cortical cataract) and advanced senile nuclear cataractous lenses. There was no significant difference in the content of reactive amino groups in control and senile nuclear cataractous lenses. The great majority, if not all, of the epsilon-amino groups of lysine residues in lens protein exist in the free form. There was no detectable difference in the protein sulfhydryl content of control and advanced senile nuclear cataractous lens proteins after reduction with mercaptoethanol.
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The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH4) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH4), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in Vmax. At pH 7.0, phosphorylation caused increased activation of TH by increasing Vmax as well as reducing the Km for cofactor. The K1 for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in Km for tyrosine at any pH or with any cofactor studied, although the Km for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH4 than with BH4 as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.
Two metabolites, 4-hydroxyisovaleric acid and mesaconic acid, have been identified and quantified in the urine of a patient with isovaleric acidemia. These compounds do not appear to have been reported previously as being components of human metabolism. In addition, large quantities of 3-methylbutyrolactone, the lactone of 4-hydroxyisovaleric acid, were observed in the volatile profile obtained by headspace chromatography. The demonstration of 4-hydroxyisovaleric acid supports the contention that urinary methylsuccinic acid seen in patients with isovaleric acidemia has arisen by omega-oxidation of isovaleric acid. The identification of mesaconic acid may indicate that the methylsuccinic acid formed in these patients is subject to further metabolism.
Two unusual sulphur-containing amino acids have been isolated from urine of a baby who died with major physical malformations and failure of growth and development. Sensitive mass spectrometric methods were used to identify the nanomole quantities of the compounds available as S-(2-carboxypropyl)-cysteine and S-(2-carboxypropyl)-cysteamine. Incubation of fibroblasts in either [14C]Valine or [35S]cysteine resulted in radioactive labelling of the compounds, suggesting their origin from conjugation of methacrylic acid with cysteine and subsequent decarboxylation of the cysteine conjugate. Specific assay of methacrylyl-CoA hydratase is needed for final proof that this is a new inborn error of that enzyme, but these findings and parental consanguinity make this very likely. It seems possible that methacrylic acid or one of its derivatives may have caused the malformations present in the baby.
Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C