Norbert W. Seidler

Exercise causes oxidative damage to rat skeletal muscle microsomes while increasing cellular sulfhydryls

The physiological and biochemical demands on contracting muscle make this tissue particularly susceptible to molecular and cellular damage. We looked at membrane structures in cardiac and skeletal muscle and in erythrocytes for exercise-induced lipid peroxidation. These tissues were removed from each of the rats used in this study. We also examined and compared the effects of exercise on the redox status of blood plasma, erythrocytes and cardiac and skeletal muscle from the same rats. We used a swim stress protocol to exercise the rats to exhaustion. Some form of chemical modification or oxidative damage to membranes was observed in all of the tissues tested. Cardiac muscle microsomes from exercised rats exhibited increased malondialdehyde and decreased phospholipid (control, 249.1 vs exercised, 120.6 nmols phospholipid/mg protein). Skeletal muscle microsomes showed decreased sulfhydryls, decreased phospholipid (control, 1,276.9 vs exercised, 137.7 nmols phospholipid/mg protein), increased malondialdehyde and greater protein crosslinking after exercise. Erythrocyte membranes also exhibited exercised-induced protein oxidation. However, the total cellular sulfhydryl content remained the same in erythrocytes and cardiac tissue but increased in blood plasma (control, 10.8 vs exercised, 24.7 mumols SH/dl plasma) and skeletal muscle after exercise. We conclude that exercise profoundly effects membrane structures. The body compensates for this lipid peroxidation and protein damage by increasing total cellular sulfhydryls in blood plasma and skeletal muscle which would aid in repair of the damaged membranes.

Effects of thermal denaturation on protein glycation.

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosines ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosines anti-inflammatory activity may be due in part to carnosines ability to prevent glycation of denatured protein.

Methylglyoxal-induced glycation affects protein topography

Methylglyoxal is a metabolic byproduct that is elevated in diabetic tissue. We examined the effects of methylglyoxal on cytosolic aspartate aminotransferase (cAAT), which is an enzyme previously shown to be modified by glyceraldehyde, acrolein, and ribose 5-phosphate. In the present study we observed that methylglyoxal caused real-time changes in tryptophan (intrinsic) fluorescence. Millimolar concentrations of methylglyoxal predominately decreased the fluorescence emission at 388 nm. While micromolar concentrations also decreased emission at 388 nm, low levels of methylglyoxal caused a prominent redshift in the wavelength of maximal emission. The changes in intrinsic fluorescence reflect definable changes in protein topography. These observations are consistent with a change in conformation that is more compact than that of native cAAT, suggesting that intramolecular cross-links (i.e., lysine-lysine) or hydrophobic pockets (i.e., carboxyethyl-lysines) were formed. Methylglyoxal also inhibited activity, and the inhibition correlated with the methylglyoxal-induced change in protein conformation.

Carnosine prevents the glycation-induced changes in electrophoretic mobility of aspartate aminotransferase

Carbohydrate‐derived aldehydes cause irreversible loss of protein function via glycation. We previously observed that glyceraldehyde 3‐phosphate (Glyc3P) abolishes the enzyme activity of cardiac aspartate aminotransferase (cAAT). We also examined the protective effects of carnosine against Glyc3P‐induced loss of enzyme activity. The present study looked at carnosine’s prevention of Glyc3P‐induced change in protein structure. Purified cAAT (2 mg protein/mL) was incubated with various concentrations of carnosine (1–20 mM) in the presence of Glyc3P (500 μM) for 4 days at 37ºC. Following incubation, samples were analyzed by SDS‐polyacrylamide gel electrophoresis. Carnosine showed prevention of protein modification at carnosine‐to‐Glyc3P ratios of 10:1 or greater. There was a progressive loss of the unmodified cAAT protein band as Glyc3P concentration was increased. Additionally, the gel position of the Glyc3P‐modified cAAT protein varied over time. The apparent molecular weight (MWapp) of the Glyc3P‐modified cAAT protein that formed after 1 day at 37ºC (500 μM) was greater than its MWapp after 2 days, suggesting that a chemical rearrangement of the initial adduct occurs. These observations support the hypothesis that carnosine is an antiglycation agent and that its mechanism of action involves prevention of protein modification.

Carnosine prevents glyceraldehyde 3-phosphate-mediated inhibition of aspartate aminotransferase.

Abstract Post-mitotic tissues, such as the heart, exhibit high concentrations (20u2009mM) of carnosine (β-alanyl-l-histidine). Carnosine may have aldehyde scavenging properties. We tested this hypothesis by examining its protective effects against inhibition of enzyme activity by glyceraldehyde 3-phosphate (Glyc3P). Glyc3P is a potentially toxic triose; Glyc3P inhibits the cardiac aspartate aminotransferase (cAAT) by non-enzymatic glycosylation (or glycation) of the protein. cAAT requires pyridoxal 5-phosphate (PyP) for catalysis. We observed that carnosine (20u2009mM) completely prevents the inhibition of cAAT activity by Glyc3P (5u2009mM) after brief incubation (30u2009min at 37u2009°C). After a prolonged incubation (3.25u2009h) of cAAT with Glyc3P (0.5u2009mM) at 37u2009°C, the protection by carnosine (20u2009mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95u2009±u200918.2u2009μmol/min per mg protein (mean ±u2009SD), minus carnosine and 100u2009±u20092.4, plus carnosine; control activity was 172u2009±u20093.9. When PyP (1.0u2009μM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93u2009±u200914.8, minus carnosine and 151u2009±u200916.8, plus carnosine, Pu2009<u20090.001; control activity was 180u2009±u200917.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger.

Carnosine promotes the heat denaturation of glycated protein

Glycation alters protein structure and decreases biological activity. Glycated proteins, which accumulate in affected tissue, are reliable markers of disease. Carnosine, which prevents glycation, may also play a role in the disposal of glycated protein. Carnosinylation tags glycated proteins for cell removal. Since thermostability determines cell turnover of proteins, the present study examined carnosines effect on thermal denaturation of glycated protein using cytosolic aspartate aminotransferase (cAAT). Glycated cAAT (500 microM glyceraldehyde for 72h at 37 degrees C) increased the T(0.5) (temperature at which 50% denaturation occurs) and the Gibbs free energy barrier (DeltaG) for denaturation. The enthalpy of denaturation (DeltaH) for glycated cAAT was also higher than that for unmodified cAAT, suggesting that glycation changes the water accessible surface. Carnosine enhanced the thermal unfolding of glycated cAAT as evidenced by a decreased T(0.5) and a lowered Gibbs free energy barrier. Additionally, carnosine decreased the enthalpy of denaturation, suggesting that carnosine may promote hydration during heat denaturation of glycated protein.

Non-Enzymatic Glycosylation (or Glycation) and Inhibition of the Pig Heart Cytosolic Aspartate Aminotransferase by Glyceraldehyde 3-Phosphate

Glyceraldehyde 3-phosphate (Glyc3P), a glycolytic intermediate, non-enzymatically glycosylated (or glycated) and inhibited the pig heart cytoplasmic aspartate aminotransferase (cAAT). Glyc3P (5.0 mM) decreased cAAT activity by 47% after 1 min at 23 degrees C. cAAT activity remained unchanged after a 24 h incubation with either glucose 6-phosphate (5.0 mM) or ribose 5-phosphate (5.0 mM). Increasing the incubation pH from 6.4 to 7.8 or the incubation temperature from 23 degrees C to 50 degrees C enhanced Glyc3Ps inhibitory effect on cAAT activity. Glyc3P (250-500 µM) decreased the thermal stability of cAAT as evidenced by lowering the T(m) or temperature that caused a 50% irreversible loss of cAAT activity (69 degrees C, control; 58.5 degrees C, 500 µM Glyc3P). Glyc3P decreased cAAT amino group content and increased glycation products, which were measured by adduct formation, fluorescence and protein crosslinking.

Acrolein modifies and inhibits cytosolic aspartate aminotransferase.

Acrolein is a reactive lipid peroxidation byproduct, which is found in ischemic tissue. We examined the effects of acrolein on cytosolic aspartate aminotransferase (cAAT), which is an enzyme that was previously shown to be inhibited by glycating agents. cAAT is thought to protect against ischemic injury. We observed that acrolein cross-linked cAAT subunits as evidenced by the presence of high molecular weight bands following SDS-PAGE. Acrolein-modified cAAT resisted thermal denaturation when compared with native cAAT. We also observed a decrease in intrinsic fluorescence (290 nm, ex; 380 nm, em). These observations are consistent with an acrolein-induced change in conformation that is more rigid and compact than native cAAT, suggesting that intramolecular cross-links occurred. Acrolein also inhibited activity, and the inhibition of enzyme activity correlated with the acrolein-induced formation of cAAT cross-links.

INHIBITION OF THE CARDIAC SARCOPLASMIC RETICULUM Ca2+-ATPASE BY GLUCOSE 6-PHOSPHATE IS Ca2+ DEPENDENT

Defects in the structure or function of the cardiac sarcoplasmic reticulum (CSR) Ca2+-ATPase presumably contribute to the Ca2+ imbalance in the diabetic myocardium. The susceptibility to nonenzymatic protein glycation by glucose metabolites is suggested due to the relatively high percent of target lysines and arginines (approaching 15 mol%) at the ATP binding and phosphorylation domains. Brief incubations (15 min) of CSR microsomes at 24 degrees C in the presence of 5.0 mM glucose 6-phosphate (Glc6P) inhibited Ca2+-dependent ATPase maximal activity relative to controls. Inhibition was only observed when incubations contained 0.1 mM CaCl2 (1.86 micromol ATP hydrolyzed x mg-1 x min-1, +Glc6P versus 2.78, control). Nonconvergent regression lines drawn from maximal velocities as a function of CSR microsome concentration indicate an irreversible mechanism of inhibition which is supported by an observed depletion in CSR amine content (2.98 micromol -NH2 groups/mg microsomal protein, +Glc6P versus 3.34, control). Glucose 6-phosphate (5.0 mM) in Ca2+-free incubations (plus 0.1 mM EGTA) had no affect on either enzyme activity or total amine content. These data suggest that the E1 but not the E2 conformation of the CSR Ca2+-ATPase is susceptible to Glc6P-mediated modification resulting in diminished maximal Ca2+-dependent ATPase activity.

Methionine Metabolism: A Window on Carcinogenesis?

Recent experimental evidence links changes in methionine metabolism to the onset and progression of cancer. Aberrant methylation reactions and polyamine synthesis may alter genome stability, gene expression, and cell proliferation.