Chemiluminescent standards for quantitative comparison of two-dimensional electrophoresis western blots.

Chemiluminescent probes offer highly sensitive quantitative analyses of proteins blotted from electrophoretic gels onto a supporting matrix (e.g. nitrocellulose or polyvinylidene difluoride). Visualization of signals from probes involves the emission of light that is dependent on a number of variables (e.g. conjugated enzyme activity, antibody titer, hybridization efficiency, substrate concentration, chemical reactivity, temperature, etc.). Thus, it is important to be able to correct for these variations. For example, the exposure time of the blot to the detection medium (e.g., film or digital camera) is a critical variable in the final results. Several protein samples separated on a single blot (e.g. one-dimensional resolution) can be compared from the ratio of the individual proteins, but comparison of separate blots completed on different days requires a chemiluminescent standard. The situation is more complex when only one sample per gel/blot is used (i.e. two-dimensional electrophoresis (2-DE)). This paper describes a method for preparing agarose embedded standardized strips that contain dilutions of antigens that can be visualized with the corresponding probe antibody. This standardization strip can be produced from common laboratory supplies and provides a method to correct for alterations in chemiluminescent intensities from different 2-DE analysis. Several standardization strips can be produced simultaneously and then used during the electroblotting step of different blots on different days.

Identification of protein carbonyls after two-dimensional electrophoresis.

The oxidative modification of proteins plays a major role in a number of human diseases, but identity of the specific proteins that are most susceptible to oxidation has posed a difficult problem. Protein carbonyls are increased after oxidative stress, and after derivatization with 2,4-dinitrophenyl hydrazine (DNP) they can be detected by various analytical and immunological methods. Although high resolution two-dimensional electrophoresis (2-DE) can resolve virtually all proteins present in a cell or tissue it has been difficult to determine the oxidized proteins because the DNP-derivatization process alters the isoelectric points of proteins, and additional procedures must be utilized to remove reaction byproducts. These additional procedures can lead to loss of sample, and poor isoelectric resolution on immobilized pH gradient (IPG) strips. We have developed a method that allows the IPG strips to be derivatized with DNP directly following isoelectric focusing of the proteins. This method allows the visualization of oxidized proteins by 2-DE with high reproducibility.

Oral polymeric N-acetyl-D-glucosamine and osteoarthritis.

Many patients with arthritis are using alternative modes of therapy, including nutritional supplements, to treat their arthritis. Most patients never tell their doctors that they are taking alternative medications, and few doctors even ask about such activities. Over-the-counter supplements are expensive and consume large amounts of patients' healthcare dollars. Glucosamine has been widely touted as being an effective arthritis treatment. The authors designed and undertook a study to test the efficacy of a polymer of N-acetyl-D-glucosamine (NAG), or POLY-Nag, in a double-blind, placebo-controlled study in patients with osteoarthritis. Results indicate that POLY-Nag may be useful in treating patients with osteoarthritis.

Oxidized proteins in Alzheimer's plasma.

The levels of oxidatively modified proteins were examined in blood from Alzheimer's disease (AD) patients, non-AD controls, and AD relatives. Oxidative modification was measured by reacting the protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH). The total oxidized proteins were determined by HPLC, while specific protein oxidation was assessed from Western blots of electrophoretic gels using antibody to the DNP derivatives. Statistically significant elevations (P < 0.05) of total oxidized proteins were observed in both AD subjects and AD relatives when compared with non-AD controls. Moreover, a protein band (e.g., MW = 78-kDa) was uniquely oxidized in the plasma of AD subjects. Furthermore, this protein from AD subjects was more susceptible to in vitro oxidation. These data suggest that such oxidized proteins may be useful as biomarkers for the detection and evaluation of AD.

Age-related permeability changes in rabbit corneas.

The present study was designed to determine whether the corneal penetration of test compounds is altered in aging. Experiments were carried out by means of passive transport under steady-state conditions using in vitro diffusion cells. Permeabilities of a variety of compounds with different physicochemical properties were measured in young (six weeks) and old (three to four years) intact and denuded (wounded) rabbit corneas. There was a marked difference in penetration of compounds between young and aged corneas. A significant decrease in permeability with age was observed. The degree of difference depended on the lipophilicity and molecular weight of the compound and the integrity of the epithelial cell layer. The difference was more pronounced for large hydrophilic than small lipophilic compounds in the intact corneas. The difference in permeabilities of test compounds between young and old denuded corneas was essentially the same (about 2-fold). These studies provide clues to the fundamental biochemical difference in young and old corneas and better enables the development of rationales for efficient drug and nutrient delivery across the cornea.

Reactive oxygen species: the unavoidable environmental insult?

Reactive oxygen species (ROS) are generated by a variety of sources from the environment (e.g., photo-oxidations and emissions) and normal cellular functions (e.g., mitochondrial metabolism and neutrophil activation). ROS include free radicals (e.g., superoxide and hydroxyl radicals), nonradical oxygen species (e.g., hydrogen peroxide and peroxynitrite) and reactive lipids and carbohydrates (e. g., ketoaldehydes, hydroxynonenal). Oxidative damage to DNA can occur by many routes including the oxidative modification of the nucleotide bases, sugars, or by forming crosslinks. Such modifications can lead to mutations, pathologies, cellular aging and death. Oxidation of proteins appears to play a causative role in many chronic diseases of aging including cataractogenesis, rheumatoid arthritis, and various neurodegenerative diseases including Alzheimer's Disease (AD). Our goal is to elucidate the mechanism(s) by which oxidative modification results in the disease. These studies have shown that (a) cells from old individuals are more susceptible to oxidative damage than cells from young donors; (b) oxidative protein modification is not random; (c) some of the damage can be prevented by antioxidants, but there is an age-dependent difference; and (d) an age-related impairment of recognition and destruction of modified proteins exists. It is believed that mechanistic insight into oxidative damage will allow prevention or intervention such that these insults are not inevitable. Our studies are also designed to identify the proteins which are most susceptible to ROS damage and to use these as potential biomarkers for the early diagnosis of diseases such as AD. For example, separation of proteins from cells or tissues on one- and two-dimensional gels followed by staining for both total protein and specifically oxidized residues (e.g., nitrotyrosine) may allow identification of biomarkers for AD.

Effect of hyperbaric oxygen on human skin cells in culture and in human dermal and skin equivalents.

A critical stage of cutaneous wound healing is the development and maturation of the epidermis. In the aged, and in certain pathologies, this repair process is compromised due to a variety of deficiencies, one of which is tissue oxygenation. Several phases of wound healing are dependent on adequate tissue oxygen levels, and hyperbaric oxygenation has been shown to transiently elevate these levels. The use of human cell monolayers, dermal equivalents and human skin equivalents provide excellent opportunities for studying wound healing using in vivo relevant models. The goal of this study was to examine the effect of hyperbaric oxygen on cell proliferation, differentiation, and matrix biosynthesis in monolayer cultures and epidermopoiesis in the developing skin equivalent. Normal human dermal fibroblasts, keratinocytes and melanocytes, dermal equivalents and skin equivalents were exposed to hyperbaric oxygen at pressures up to three atmospheres, for up to 10 consecutive daily treatments lasting 90 minutes each. Increase in fibroblast proliferation (cf., 30% at 1 atmosphere after 10 days treatment), was observed without a significant effect on proliferation of normal human melanocytes and glycosaminoglycan synthesis. Stimulation of collagen synthesis after two days of treatment was only significant at 1 atmosphere (about 20% increase) but this differential was not observed after 5 days of treatment. Hyperbaric oxygenation above 2 atmospheres, inhibited proliferation of fibroblasts and keratinocytes in cell monolayer cultures (e.g., a 10 day treatment at 3 atmospheres appeared cytostatic to keratinocytes). In contrast, hyperbaric treatment up to 3 atmospheres dramatically enhanced keratinocyte differentiation, and epidermopoiesis in the complete human skin equivalent. These results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue. The results also show the utility of the human skin equivalent as a model for evaluation of parameters involved in wound healing.

A double stain for total and oxidized proteins from two-dimensional fingerprints.

Oxidative modification of proteins plays a major role in the etiology of aging and age-related diseases. For example, in Alzheimer's disease, although evidence points to oxidation of proteins as a causative factor in loss of cognitive abilities, it is not known which specific proteins of the brain are most susceptible to these modifications. Thus, it is of interest to identify the specific proteins which are susceptible to oxidation in vivo. Two-dimensional protein fingerprint methods offer the analytical potential for resolution of thousands of individual proteins from tissues, and the oxidized proteins can be visualized with immunological probes. Sensitive methods permit recovery and sufficient amino acid sequencing to identify these proteins. However, for such analyses it is essential to simultaneously analyze both protein content and level of oxidation. We have evaluated several approaches, identified the sources of artifacts and interferences, and developed a double-staining procedure that allows visualization and quantitation of total protein patterns as well as the specific oxidized proteins from two-dimensional protein fingerprints. The method has been applied to cells grown in culture and to tissue extracts from young and old animals.

Molecular wear and tear leads to terminal marking and the unstable isoforms of aging.

Our studies focus on the mechanisms of molecular wear and tear, terminal marking, protein degradation, and how these processes are altered with age. Molecular wear and tear directly links catalysis with postsynthetic terminal marking. For example, the binding of ligands and catalysis cause conformational changes that are transmitted from the catalytic center to the site of terminal marking and enhance the rates of specific covalent modifications, such as deamidation or oxidation. These oxidations or deamidations can introduce "KFERQ motifs" into proteins, which may permit them to be recognized and transported to the site(s) of complete degradation. Terminally marked proteins accumulate in aging cells and tissues and account for many of the health problems of the elderly. Two-dimensional protein fingerprinting coupled with immunostaining permits identification and characterization of these proteins. Free-radical traps or caloric restriction, which may prevent the formation or enhance the degradation of terminally marked proteins, may be useful in the prevention or treatment of age-associated health problems, including dementia.

Effect of active-site modification on the terminal marking deamidation of triosephosphate isomerase.

The conformational change which results from the opening and closing of the hinged lid over the catalytic center of triosephosphate isomerase is transmitted to the subunit interface of the dimer and eventually leads to the spontaneous, specific deamidation of Asn71. This is followed by the deamidation of Asn15 approximately 5 A away on the neighboring subunit and leads to destabilization of the protein and degradation. However, it has not been established whether this molecular wear and tear occurs via an intra-subunit or inter-subunit transmission of the conformational change. We have studied this first step in the terminal marking by reacting the active-site Glu165 with the substrate analog 3-chloroacetol phosphate (CAP) which immobilizes the hinged lid. Under mild deamidation conditions (pH 9.5, 24 h, 30 degrees C) the native homodimer readily deamidated. In contrast, the CAP-modified homodimers were resistant to deamidation. Heterodimers composed of one native subunit and one CAP subunit showed intermediate deamidation. When the native and CAP-labeled subunits were resolved by electrophoresis in urea gels, it was found that the unlabeled subunit had preferentially deamidated. These data coupled with molecular modeling considerations show that restricting movement of the hinged lid prevents deamidation of Asn71 on that subunit, but that the other subunit with the free hinged lid functions independently and still deamidates. Thus, the conformationally induced wear and tear appears to be largely intra-subunit. These observations clearly link catalysis with terminal marking of the protein and suggest a general mechanism for how other enzymes may wear out.

The 28K protein in urinary bladder, squamous metaplasia and urine is triosephosphate isomerase.

OBJECTIVES: The objective of this study was to establish the identity of a protein found in high concentrations in squamous metaplasia of the bladder. DESIGN AND METHODS: The protein was isolated and subjected to a series of physical, chemical, and catalytic studies. RESULTS: In the normal urothelium the protein was confined to a juxtanuclear pattern on the luminal side of the umbrella cells; in squamous metaplasia and squamous cell carcinoma the protein was increased and exhibited a more diffuse intracellular distribution. The protein was found to be identical to triosephosphate isomerase (EC 5.3.1.1; TPI) with respect to its immunological properties, native and subunit molecular weights, electrophoretic mobility, catalytic activity, and amino acid sequence. CONCLUSIONS: While the basis for the altered distribution of TPI remains to be established, the increased amounts of the protein in urine or bladder tissue may be indicative of squamous metaplasia, squamous cell carcinoma, or other bladder injuries.

Pilot study of oral polymeric N-acetyl-D-glucosamine as a potential treatment for patients with osteoarthritis.

Glucosamine and its derivatives, such as glucosamine sulfate and N-acetyl-D-glucosamine (NAG), have been shown to be effective in the treatment of patients with osteoarthritis. Unfortunately, the half-life of glucosamine in the blood is relatively short; therefore, a sustained-release form of the compound would be highly desirable. The purpose of this pilot study was to determine whether the polymeric form of NAG (POLY-Nag) could provide a longer-lasting oral source of NAG. Ten healthy subjects each ingested 1 g/d of either NAG or POLY-Nag for 3 days. After a 4-day washout period, each subject was crossed over to receive the other compound for 3 days. Serum samples were collected and analyzed using high-performance liquid chromatography. Results show that orally ingested NAG and POLY-Nag are absorbed, resulting in increased serum levels of NAG, and POLY-Nag appears to be at least as effective as NAG. Serum levels of NAG had decreased by 48 hours after cessation of ingestion of NAG or POLY-Nag but were still above baseline levels. Increases in serum glucosamine levels indicate that NAG and POLY-Nag are converted to glucosamine in vivo. In conclusion, POLY-Nag may provide a source of serum glucosamine for treatment of patients with osteoarthritis. Longer and more rigorous pharmaco-kinetic and clinical studies need to be done.

Terminal marking of triosephosphate isomerase: consequences of deamidation.

Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.

Terminal marking of avian triosephosphate isomerases by deamidation and oxidation.

Triosephosphate isomerase (TPI) provides an excellent model for terminal marking and protein degradation. Mammalian TPI is terminally modified by deamidation at Asn71-Gly, resulting in unfolding, dissociation, and proteolysis. In contrast, chicken TPI, which contains a lysine at position 71, is terminally modified by the oxidation of Cys126. Thus, both deamidation and oxidation initiate degradation of TPI from different species. To explore the terminal marking in other avians, we have purified the turkey TPI to homogeneity and determined its characteristics. Although the molecular properties of the turkey and chicken TPI were very similar, their tolerances to temperature, oxidants, and alkaline pH were very different. For example, chicken TPI was inactivated 80% in either 10 mM oxidized glutathione or H2O2, whereas 120 mM GSSG had no effect on turkey TPI, and > 120 mM H2O2 was needed for 80% inactivation. Under alkaline conditions that cause rapid deamidation of the mammalian TPI, neither avian enzyme deamidated. Chicken TPI, however, aggregated. Aggregation was reversed by 2-mercaptoethanol. Under prolonged exposure to milder conditions the turkey enzyme was completely inactivated and deamidated at Asn15-Gly. Thus, there are marked differences in the susceptibility of these two avian enzymes to oxidation and deamidation, and their terminal marking mechanisms appear to be different.

The culture of rat lenses in high sugar media: effect on mixed disulfide levels.

Lens proteins are long lived proteins with those in the center of the lens predating the birth of the individual. As a result, they are subject to a host of modifications and damage through a variety of mechanisms. Two such modifications have been proposed as primary events which could cause conformational changes potentiating further modifications. These are non-enzymatic glycation and mixed disulfide formation. Human lenses accumulate protein-thiol mixed disulfides of three kinds throughout the lifespan. The presence of one of these, protein-glutathione (PSSG) mixed disulfide has been shown to be intimately involved in protein aggregation. We have utilized ex vivo lens culture and in vitro incubations of purified gamma-crystallin to evaluate the following hypotheses. A) Lenses cultured with a high sugar media will form higher mixed disulfide levels than controls; B) glycation of lens proteins will be dependent on initial mixed disulfide level. Xylose levels in the cultured lens rise rapidly (to 23 mM by 4 h), and the level of glycation after one week is elevated 6-7% over control values. Mixed disulfide levels are also substantially increased but not more than for lenses cultured in control media. gamma-Crystallin modified with 0, 1, or 5 equivalents of GSH was differentially glycated by radioactive fructose. The amount of fructose bound by the protein was found to be inversely related to the extent of mixed disulfide formation. These results indicate that 1) protein modification of one kind may influence further modifications of other types; 2) glycation of lens proteins has no effect on mixed disulfide formation in this system; 3) the sulfhydryl status of lens proteins can affect the potential for protein glycation.

Ascorbic acid mediated alteration of alpha-crystallin secondary structure.

Glycation, the non-enzymatic addition of sugar or other carbonyl compounds to the amino groups of a protein, has been shown to occur with a variety of sugars and a diverse group of proteins. This type of alteration is believed to be an important component of aging for lens proteins and perhaps in cataractogenesis. Glycation has been shown to alter function and spectroscopic techniques have shown that in many cases conformational changes have occurred. Circular dichroism spectroscopy has documented modifications to alpha-crystallin tertiary structure induced by glucose and glucose 6-phosphate but generally no change to secondary structure. Ascorbate and is oxidized derivative dehydroascorbate have been shown to be powerful glycating agents as well as forming cross-links between peptide chains. In this study, alpha-crystallin incubated with ascorbic acid for one or two wk shows significant incorporation of ascorbate, non-reducible cross-links between the protein chains and altered CD spectra in the far UV region indicative of secondary structure modification.

Deamidation of triosephosphate isomerase in reverse micelles: effects of water on catalysis and molecular wear and tear.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

The hinged lid of yeast triose-phosphate isomerase. Determination of the energy barrier between the two conformations.

Covalent modification of Glu165 in the catalytic center of triose-phosphate isomerase with the substrate analogue 3-chloroacetol phosphate traps the complex in two conformations. The two resulting 31P NMR resonances at 6.9 and 5.7 ppm appear to reflect conformations in which the hinged lid (residues 167-176) is in the open and closed positions. The conformation represented by the 5.7-ppm resonance is more stable, and unfolding and refolding in guanidine converts all of the molecules to the 5.7-ppm conformation. The complete conformational transition from 6.9 to 5.7 ppm also takes place as a function of time and temperature. Under these conditions the native enzyme retains more than 80% of the catalytic activity, indicating that this conversion is not due to thermal denaturation of the enzyme. Circular dichroic and fluorescence spectroscopy indicate that the 3-chloroacetol phosphate-modified enzyme does not undergo major structural changes. From the temperature dependence of this transition, an energy barrier of 144 kJ/mol (34.4 kcal/mol) was calculated for this conversion.

Limited proteolysis of triose-phosphate isomerase and characterization of the catalytically active peptide complex.

Limited proteolysis of the triose-phosphate isomerase (EC 5.3.1.1) by subtilisin generates peptides that remain noncovalently attached and catalytically active. Edman degradation of the peptides showed that the primary proteolytic sites for yeast triose-phosphate isomerase are the Leu174-Ala175 bond followed by Ser52-Leu53. The Leu174-Ala175 site is of particular interest, since it forms part of the hinged lid that closes over the catalytic center, and this bond is only 12.2 A (open) or 9.8 A (closed) from the catalytic residue Glu165. The higher Km, kcat, and kcat/Km values exhibited by the catalytically active peptide complex suggest that the substrate is not bound as tightly to the catalytic center. In addition, increased methylglyoxal formation by the cleaved enzyme indicates that the enzyme-substrate complex is less protected from the solvent. Circular dichroic and fluorescence spectra show that the overall structure of the peptide complex is similar to the native enzyme but with local structural perturbations around the tryptophans. Also, the peptide complex is more susceptible to denaturation by guanidine and exhibits lower Tm values, indicating a loose interaction between the fragments. Unfolding, dissociation, and refolding experiments suggest that the fragments have strong inherent secondary structural features and can reassociate into catalytically active structures.

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.