Roger T. Dean

Free radicals, lipids and protein degradation

Abstract Primary oxygen radicals produced in cells and their secondary lipid radical intermediates can modify and fragment proteins. The products are often more susceptible to enzymatic hydrolysis and so radical fluxes may accelerate proteolysis inside and outside cells.

Scavenging by alginate of free radicals released by macrophages.

Failure to eradicate mucoid forms of P. aeruginosa has implicated bacterial alginate in a local evasion of host defence mechanisms within the lung of Cystic Fibrosis (CF) patients. We have found that purified bacterial alginate scavenges free radicals released by triggered macrophages as detected by lucigenin amplified chemiluminescence (CL) and reduction of cytochrome c. In agreement with this, alginate was also able to scavenge radicals generated by a chemical system (hydrogen peroxide and copper; detected by benzoate hydroxylation and chemiluminescence), and by an enzymatic system (hypoxanthine and xanthine oxidase; detected by chemiluminescence). All inhibitions were dose-related. Oxygen consumption by neutrophils (unlike that of macrophages) could be detected in a Clark electrode, and was not reduced by alginate, confirming that scavenging of radicals was responsible for the earlier observations. These data suggest that bacterial alginate by scavenging free radicals, may favour the survival of mucoid forms of P. aeruginosa, particularly in the CF lung.

Membrane proteins are critical targets in free radical mediated cytolysis.

The hypothesis that proteins are critical targets in free radical mediated cytolysis was tested using U937 mononuclear phagocytes as targets and iron together with hydrogen peroxide to generate radicals. Those conditions which, after a lag of approx. 30 min, led to drastic lysis were also associated with very rapid membrane depolarisation. Conversely, when the early membrane depolarisation was prevented (by the addition of chelator and catalase), so was lysis. A similar correlation between early membrane depolarisation and subsequent lysis was also observed when the cells were exposed to a toxin from Actinobacillus actinomycetemcomitans. Those conditions of radical attack which led to lysis normally caused substantial lipid peroxidation. However, depolarisation and subsequent lysis were not prevented even when lipid peroxidation was completely suppressed by exogenous antioxidant. ATP levels were not grossly affected within the critical first 30 min period. These data exclude lipids and ATP as the target for lytic damage. We argue therefore that proteins are probably amongst the primary targets in cytolysis by radicals.

Radical initiated α-tocopherol depletion and lipid peroxidation in mitochondrial membranes

Abstract The relationships between antioxidant status, lipid peroxidation and membrane protein integrity have been studied in an isolated mitochondrial membrane system. Tocopherol was shown to be present in both the outer and inner membrane of normal rat liver mitochondria; 77.3 and 22.3% of the total α-tocopherol was present in the outer and inner membranes, respectively. The endogenous α-tocopherol was depleted in a time-dependent manner by low levels of ferrous iron and by irradiation in the presence or absence of ferrous iron. This antioxidant depletion was followed by the appearance of lipid hydroperoxides. Fragmentation of monoamine oxidase, an integral outer membrame protein, was observed at irradiation doses that caused both antioxidant depletion and peroxide generation.

A mechanism for accelerated degradation of intracellular proteins after limited damage by free radicals

I propose that limited free radical attack upon proteins, occurring continuously in cells, creates new N‐termini (notably aspartate and glutamate) which render the proteins more susceptible to proteolysis by the ubiquitin conjugation system. I suggest that these reactions are a significant part of the previously described ‘N‐end’ and ‘PEST’ rules, which indicate amino acid termini or sequences which tend to dictate short protein half‐lives. I also argue that the N‐end rule may apply to sequestered intracellular sites, such as mitochondria, these also being sites of radical generation.

Vitamin E protects proteins against free radical damage in lipid environments

The fragmentation of the membrane protein monoamine oxidase in submitochondrial particles was induced by defined free radicals during radiolysis and by a system dependent on hydrogen peroxide and a transition metal. By injection of alpha-tocopherol in vivo, the levels of this physiological antioxidant in the mitochondrial preparations could be elevated more than ten-fold. In both radical-generating systems the presence of high levels of alpha-tocopherol in the membrane substantially retarded the protein fragmentation, in parallel with lipid peroxidation. It is suggested that membrane-bound proteins are damaged during lipid peroxidation and that alpha-tocopherol protects cells against both types of damage.

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 -days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.

A continuous-flow automated assay for iodometric estimation of hydroperoxides

An iodometric method for the analysis of hydroperoxides has been automated to allow analysis of aqueous biological samples (containing less than 20 mg/ml protein) and lipid hydroperoxide extracts. The evolution of triiodide ions is measured spectrophotometrically at 360 nm. Dependent on the type of sample, 30-60 samples can be analyzed per hour and the system allows detection of less than 100 pmol of peroxide. The assay is linear over a range of 100 pmol to 25 nmol. The sample volume used routinely was 80 microliters.

Oxygen-centred free radicals can efficiently degrade the polypeptide of proteoglycans in whole cartilage

Bovine nasal cartilage slices, biosynthetically labelled in their proteoglycan with35SO4, were used as substrate for the attack of free radicals generated on exposure to a Co60 source (which allows study of single radical species), and by chemical and enzymatic means. Systems generating hydroxyl (OH⋅) and superoxide (02⋅-) radicals degraded the proteoglycan efficiently, while the hydroperoxy radical (HO2⋅) was less efficient ; addition of appropriate radical scavengers inhibited degradation. The radioactive products were heterogeneous in molecular size, but with doses up to 3600 Gy were the same size range as intact chondroitin sulphate. They contained free amino groups, and more were liberated by aminopeptidase M digestion, implying that at least a small peptide was present. Thus a major site of radical attack may be the polypeptide chain. We suggest that free-radical fragmentation of polypeptides may be important both in extracellular catabolism and in intracellular proteolysis.

Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: Evidence for a second mechanism of spontaneous secretion?

Abstract β-Hexosaminidase secreted by peritoneal macrophages in response to stimulation by zymosan or NH 4 Cl, or spontaneously by a macrophage-like cell line (P388D 1 ), is susceptible to receptor-mediated endocytosis by human fibroblasts. This endocytosis is almost completely blocked by exogenous mannose-6-phosphate and therefore seems to depend on a mannose-6-phosphate ligand on the enzyme. It is suggested that macrophage lysosomal enzyme packaging may involve mannose-6-phosphate recognition markers, and that a continuous hypersecretion mechanism may exist which does not depend on a defect in this ligand.