Endogenous plastic composite material in the Alzheimer's brain.

Accumulation of amyloid beta (Abeta) peptide in brain is the hallmark of Alzheimer's disease (AD). The resulting plaques though fibrous in nature may also consist of additional structures currently poorly defined. We hypothesize that plastic composite material contributes to plaque formation. This material is organized by polymers of acrolein, which is an oxidized lipid fragment found in AD. Acrolein, a 3-carbon compound, contains a carbonyl and a vinyl group that participate in polymerization via fundamental latex chemistry. The redox and surfactant properties of Abeta allow it to catalyze the polymerization of acrolein. We previously reported observations of thin plastic fragments of Abeta-polyacrolein. The current paper outlines the proposed steps in forming these plastic fragments. Endogenous plastic composite material may significantly contribute to the pathogenesis of AD.

Glycation of aspartate aminotransferase and conformational flexibility.

Glycation of proteins alters biological function and changes cellular processes. Our study investigated the conformational changes that accompany glycation using the cardiac aspartate aminotransferase (cAAT). We examined the effects of brief and prolonged exposure of cAAT to glyceraldehyde (Glyc) and ribose 5-phosphate (R5P). When cAAT was briefly incubated (3.5 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, cAAT activity and 1-anilinonaphthalene 8-sulfonate (ANS) binding increased relative to control. After prolonged incubation (64 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, activity and ANS binding decreased relative to control. Furthermore, upon prolonged incubation of cAAT with 500 microM Glyc (14.5 h) or 2 mM R5P (64.25 h) at 37 degrees C, the denaturation curves shifted to the right relative to control. We conclude that upon brief incubation with Glyc and R5P, cAAT exhibited a more open and flexible structure and upon prolonged incubation, a more rigid structure.

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 24h 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 Glyc3P's inhibitory effect on cAAT activity. Glyc3P (250-500 microM) decreased the thermal stability of cAAT as evidenced by lowering the Tm or temperature that caused a 50% irreversible loss of cAAT activity (69 degrees C, control; 58.5 degrees C, 500 microM Glyc3P). Glyc3P decreased cAAT amino group content and increased glycation products, which were measured by adduct formation, fluorescence and protein crosslinking.

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 microM) for 4 days at 37 degrees C. 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 37 degrees C (500 microM) 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.

Post-mitotic tissues, such as the heart, exhibit high concentrations (20 mM) of carnosine (beta-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 (20 mM) completely prevents the inhibition of cAAT activity by Glyc3P (5 mM) after brief incubation (30 min at 37 degrees C). After a prolonged incubation (3.25 h) of cAAT with Glyc3P (0.5 mM) at 37 degrees C, the protection by carnosine (20 mM) persisted but PyP availability was affected. In the absence of PyP from the assay medium, cAAT activities (plus Glyc3P) were 95 +/- 18.2 micromol/min per mg protein (mean +/- SD), minus carnosine and 100 +/- 2.4, plus carnosine; control activity was 172 +/- 3.9. When PyP (1.0 microM) was included in the assay medium, cAAT activities (plus Glyc3P) were 93 +/- 14.8, minus carnosine and 151 +/- 16.8, plus carnosine, P < 0. 001; control activity was 180 +/- 17.7. These data, which showed carnosine moderating the effects of both Glyc3P and PyP, suggest that carnosine may be an endogenous aldehyde scavenger.

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.

Diabetic cardiomyopathy: the significance of creatine.

Diabetics exhibit a greater incidence of cardiovascular disease than non-diabetics. The vascular changes that occur are well documented and are thought to promote other clinical manifestations such as cardiomyopathy. Research has shown that the pathogenic events in the myocyte may occur independently of atherosclerotic processes. The atherosclerotic changes in diabetes involve non-enzymatic glycation of extracellular basement membrane proteins. We hypothesize that intracellular glycation events occur in cardiac tissue that alter intermediary metabolism, particularly Ca2+ homeostasis, which leads to cell dysfunction. Additionally, we hypothesize that the high steady state intracellular concentrations of unphosphorylated creatine may offer protection against the formation of advanced glycation endproducts by reacting directly with glucose metabolites that may have reached toxic levels in the myocyte of diabetics.

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.

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.

NSAID-induced gastroenteropathy: a biochemical dissection.

The most widely accepted view is that damage to the mucosa sets the stage for lesions related to NSAIDs. An alternative hypothesis is that NSAID-induced damage to GI microvasculature is the primary pathologic event.

The role of lipid peroxidation in McArdle's disease: applications for treatment of other myopathies.

Experimental therapies for McArdle's disease have been directed toward increasing substrate availability to exercising muscle. Such therapies to date have proven largely unsuccessful. These include administration of isoproterenol to increase blood flow, glucagon treatment to elevate serum glucose and increased dietary fat intake. Each of these therapies also results in greater levels of unesterified fatty acids in blood. More recently, a high protein diet is suggested to provide increased amounts of amino acids which would be available as fuel sources. We hypothesize that the absence of myophosphorylase in McArdle's disease creates an imbalance between the enzymes of the redox systems that control the generation, propagation and inactivation of free radicals. This occurs because muscle cells are forced to rely more heavily on fatty acid oxidation. The resulting free radical damage to cellular components disrupts metabolic control and increases the permeability of membranes. Elevated levels of Ca2+ in the sarcoplasm activate proteases, phospholipases and other catabolic enzymes initiating muscle fatigue and cramping. Lipid peroxidation is a consequence of normal muscle activity and may occur unchecked in individuals with McArdle's disease. Continued muscle activity in the absence of a favorable nutritional environment may promote the progression of the disease by increasing susceptibility to oxidative stress.

The effects of pentoxifylline on the plasma membrane Ca2+ ATPase in age-separated rat and human erythrocytes.

Pentoxifylline, a dimethyl xanthine derivative given to patients with peripheral vascular disorders, increases erythrocyte deformability, diminishes Ca2+ entry, inhibits Ca(2+)-dependent transglutaminase activity and elevates ATP levels. The present study examined the effects of pentoxifylline on the Ca2+ pump ATPase, an enzyme which regulates intracellular Ca2+ levels. Studies were carried out with inside-out vesicles (IOVs) prepared from young (Ey) and old (Eo) human and rat erythrocytes. The pumping of Ca2+ depends on the concomitant hydrolysis of ATP; the two processes were measured using radiolabeled substrates. The catalytic properties of IOVs from young and old erythrocytes were stimulated by pentoxifylline when added directly to the assay medium. Pentoxifylline (0.5 to 5.0 mM) significantly activated the rates of Ca2+ dependent ATP hydrolysis in Ey and Eo IOVs of rat erythrocytes. The percent of activation was greater in the IOVs from older erythrocytes. The Ca2+ translocation was also affected by pentoxifylline. The early burst of Ca2+ uptake into IOVs decreased in the presence of pentoxifylline in Ey IOVs prepared from either species whereas steady state rates of Ca2+ transport only declined at 5.0 mM pentoxifylline. This pattern was not evident in the corresponding Eo IOVs. The response of Ey and Eo IOVs to pentoxifylline may be the basis of the difference in sensitivity of young and old erythrocytes to the drug in regulating intracellular Ca2+ concentrations.

Immunological relatedness of the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase.

The effect of anti-ATPase antibodies with epitopes near Asp-351 (PR-8), Lys-515 (PR-11) and the ATP binding domain (D12) of the Ca(2+)-ATPase of sarcoplasmic reticulum (EC 3.6.1.38) was analyzed. The PR-8 and D12 antibodies reacted freely with the Ca(2+)-ATPase in the native membrane, indicating that their epitopes are exposed on the cytoplasmic surface. Both PR-8 and D12 interfered with the crystallization of the Ca(2+)-ATPase, suggesting that their binding sites are at interfaces between ATPase molecules. PR-11 had no effect on ATPase-ATPase interactions or on the ATPase activity of sarcoplasmic reticulum. The epitope of PR-11 is suggested to be the VIDRC sequence at residues 520-525, while that of D12 at residues 670-720 of the Ca(2+)-ATPase. The use of predictive algorithms of antigenicity for identification of potential antigenic determinants in the Ca(2+)-ATPase is analyzed.

Polarized infrared attenuated total reflectance spectroscopy of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca(2+)-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl2, 1-10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing congruent to 7.5 micrograms protein/cm2, and decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH2 vibrations (2923 cm-1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm-1) of the Ca(2+)-ATPase in the Ca2-E1 state and in the EGTA and vanadate stabilized E2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH2 vibrations was not affected by changes in the concentration of KCl (25-100 mM) or Ca2+ (approximately equal to 10(-8)-10(-4) M) and by the addition of vanadate (1 mM) or Pi (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm-1) or CO stretching band (1046 cm-1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of approximately 70 degrees between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca(2+)-ATPase.

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human.

The plasma membrane Ca2+ ATPase in erythrocytes is vital for the maintenance of intracellular Ca2+ levels. Since the cytoplasmic Ca2+ concentration is elevated in older erythrocytes, the properties of the Ca2+ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca2+ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca2+ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca2+ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca2+ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca2+ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca2+ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4 degrees C). The decrease in Ca2+ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum.

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum.

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.