Degradation Of Albumin Research Articles

Cholesterol depletion by methyl-beta-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons.

We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MbetaCD. Whereas pre-incubation with MbetaCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MbetaCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.

Degradation of albumin in meningococcal sepsis

We postulate that the proteolytic degradation of albumin into fragments could link the rapidity of the shock, rash, and hypocalcaemia associated with meningococcal sepsis. We examined urine of children with meningococcal disease and urine from control children with no sepsis and found albumin fragments of about 45 kDa, 25 kDa, and less than 20 kDa only in the urine of children with meningococcal sepsis and associated purpura. Exogenous or endogenous proteases, or both, may be released in severe meningococcal sepsis and, in association with an inadequate antiprotease response, result in albumin degradation. This may be a contributory factor to the rapid shock, hypocalcaemia, and rash seen in meningococcal sepsis.

Albuminuria in patients with type 1 diabetes is directly linked to changes in the lysosome-mediated degradation of albumin during renal passage.

Previous studies by our group have shown that albumin is metabolized in rodents during renal passage and excreted in the urine as a mixture of intact protein and albumin-derived fragments. The aim of this study was to examine whether albumin is metabolized during renal passage in nondiabetic volunteers and in type 1 diabetic patients with varying levels of albuminuria. Nine nondiabetic normoalbuminuric volunteers and 11 type 1 diabetic patients with albumin excretion rates varying from normoalbuminuria to macroalbuminuria were studied. Each subject received an intravenous injection of tritium-labeled albumin ([3H]-albumin). Urine was collected at 4 h and 24 h after injection and analyzed by size exclusion chromatography. The amount of intact and fragmented albumin was quantified, and each fraction was analyzed by radioimmunoassay (RIA) for albumin. [3H]-albumin in nondiabetic volunteers was metabolized during renal passage to small peptide fragments not detectable by conventional RIA (only 0.05-3.8% of the total urinary radioactivity was associated with intact albumin). The process responsible for albumin fragmentation was similar in diabetic patients with normoalbuminuria (intact albumin represented 0.01-4.0% of total urinary radioactivity). However, there was a reduction in the fragmentation ratio (fragmented:intact) in diabetic patients with micro- or macroalbuminuria (intact albumin represented 2.7-55.5%, P = 0.048). This change in the fragmentation ratio was directly related to the degree of albuminuria. These results have important implications for understanding the mechanisms underlying albuminuria in nondiabetic volunteers and type 1 diabetic patients. In nondiabetic volunteers, the renal processing of albumin involves a relatively rapid and comprehensive degradation of albumin to small fragments (range 1-15 kDa). The degradation process is inhibited in diabetic nephropathy in proportion to the level of albuminuria detected by RIA.

The lysosomes of earthworm chloragocytes: biochemical and morphological characterization

Nine latent and sedimentable acid hydrolases have been detected in the homogenates of earthworm chloragocytes. Their full activity was revealed by treatment with Triton X-100, a Waring blendor treatment, freezing and thawing, hypotonic media or incubation at pH 5 and 25°C. Solubilization paralleled the activation of the enzymes. Together with kinetic studies, these results indicate that the acid hydrolases of the chloragocytes are inside typical lysosome-like particles whose membrane is impermeable to their substrates. It could be shown by density equilibration centrifugation that the lysosomes of those cells constitute a heterogeneous population of subcellular particles distinct from the chloragosomes. Moreover, their digestive function has been directly demonstrated by the capture and degradation of serum albumin. The lysosomes of the chloragocytes have been clearly identified as polyvesicular bodies by electron microscopic analysis of the fractions obtained by density equilibration centrifugation and by examination of the whole tissue, as such or after endocytosis of serum albumin or ferritin. Finally, our results do not support a possible relationship between the lysosomes and the chloragosomes of the chloragocytes.

ASSESSMENT OF THE DEGRADATION OF DENATURED ALBUMIN SOLDER BY HUMAN URINE

Several studies have been undertaken to evaluate the efficacy of human serum albumin (HSA) as a solder in urologic procedures. The purpose of this study was to evaluate whether albumin solder undergoes significant degradation in urine. Laser denatured 25% HSA pellets were incubated at 37C for varying times with 1 ml. of either pooled human urine or control diluent solution adjusted to the same pH and osmolality as urine. To assess the contribution of enzymatic degradation, aliquots of urine were boiled and compared with non-boiled urine and diluent. The amount of solubilized HSA in solution was measured using the Bradford assay, while the degradation of albumin was detected by SDS-PAGE electrophoresis. Approximately 5% of the albumin was degraded over a period of 7 days following incubation at 37C, regardless of treatment. SDS-PAGE revealed only minor traces of degradation in urine and controls. The very slight degradation of denatured HSA appears to be non-enzymatic, as it was observed in both urine and diluent samples. HSA solder appears to be appropriate for use in urologic reconstructive surgery since it is not appreciably degraded in the presence of urine.

Advanced glycation endproduct-specific receptors in rat and mouse osteoblast-like cells: regulation with stages of differentiation.

Advanced glycation endproducts have been implicated in the development of diabetic complications. In addition, these products could also mediate certain bone alterations such as diabetic osteopenia. Several receptors specific for advanced glycation endproduct-modified proteins have been characterized in different cell types, contributing to the recognition and degradation of senescent proteins. In the present report, we investigated the possible presence of advanced glycation endproduct-binding proteins on osteoblast-like cells. Both UMR106 and MC3T3E1 cell lines express specific advanced glycation endproduct-binding sites, with an affinity constant between 0.4 and 1.7. 10(6) M(-1), depending on the stage of osteoblastic differentiation; and with a receptor capacity of 1.5-2.0. 10(7) sites/cell. Osteoblast-like cells were also found to participate both in the uptake and degradation of advanced glycation endproduct-modified bovine serum albumin at 37 degrees C. Radiolabelled ligand blotting studies confirmed the presence of several membrane binding proteins, with apparent molecular masses of 50, 45-40, 30, 25 and 18 kDa; the major bands corresponded to 30 and 25 kDa proteins. This study provides evidence of the presence of advanced glycation endproduct-specific binding sites, and for their regulation with the stage of differentiation, in two osteoblast-like cells in culture.

Interaction between peroxide ion and phenol group which are coordinated to the same iron(III) ion

We have prepared several new iron(III) complexes with ligands which contain a phenol group; these are tetradentate [(X-phpy)H, X and H(phpy) represent the substituents on the phenol ring and N, N-bis(2-pyridylmethyl)- N-(2-hydroxybenzyl)amine, respectively] and pentadentate ligands [(R-enph-X)H; R=ethyl(Et) or methyl(Me) derivative and H(Me-enph) denotes N, N-bis(2-pyridylmethyl)- N″-methyl- N″-(2″-hydroxyl-benzylamine)ethylenediamine] and have determined the crystal structures of Fe(phpy)Cl 2, Fe(5-NO 2-phpy)Cl 2, and Fe(Me-enph)ClPF 6, which are of a mononuclear six-coordinate iron(III) complex with coordination of one or two chloride ion(s). These compounds are highly colored (dark violet) due to the coordination of phenol group to an iron(III) atom. When hydrogen peroxide was added to the solution of the iron(III) complex, a color change occurs with bleaching of the violet color, indicating that oxidative degradation of the phenol moiety occurred in the ligand system. The bleaching of the violet color was also observed by the addition of t-butylhydroperoxide. The rate of the disappearance of the violet color is highly dependent on the substituent on the phenol ring; introduction of an electron-withdrawing group in the phenol ring decreases the rate of bleaching, suggesting that disappearance of the violet band should be due to a chemical reaction between the phenol group and a peroxide adduct of the iron(III) species with an η 1-coordination mode and that in this reaction the peroxide adduct acts as an electrophile towards phenol ring. The intramolecular interaction between the phenol moiety and an iron(III)-peroxide adduct may induce activation of the peroxide ion, and this was supported by several facts that the solution containing an iron(III) complex and hydrogen peroxide exhibits high activities for degradation of nucleosides and albumin.

Degradation of transferrin and albumin by radical reactions in human plasma evaluated by immunoblot.

Immunoblot analysis showed that transferrin and human serum albumin were degraded in a similar rate on the basis of the protein weight by radical reactions using a solution containing each protein initiated by Cu2+ and hydrogen peroxide. When plasma was treated with Cu2+ and hydrogen peroxide, the rate profile of the degradation of transferrin was similar to that of albumin based on immunoblot analysis in spite that the content of these proteins in plasma was considerably different. These results suggest that highly reactive hydroxyl radicals attack these proteins indiscriminately. Present observations demonstrate that immunoblot analysis is an effective method to analyze radical reactions of each protein in the presence of many other proteins like plasma.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria

1. Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2. Four-week male Sprague-Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer=4+/-2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer=17+/-5%). 3. This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular 'shunt' pathway.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria

1.Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2.Four-week male Sprague–Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer = 4±2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer = 17±5%). 3.This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular ‘shunt' pathway.

Contribution of a peroxide adduct of copper(II)-peptide complex to modify the secondary structure of albumin.

We have found that copper(II) compounds containing a peptide group in the chelate exhibit high activity for modification or degradation of albumin in the presence of hydrogen peroxide, whereas no activity was detected for the copper(II) compounds without an amide-group. It is suggested that presence of the amide-group in the ligand may play an important role in the formation of a peroxide adduct and in activation of the peroxide ion, leading to cleavage of the peptide bond of a neighboring protein. It is implied that conversion of normal cellular prion protein PrPC into a disease-causing isoform, PrPSc is attributed to the activated peroxide ion coordinated to a copper(II) captured in the NH2-terminal domain of the PrPC.

Degradation of serum albumin by rat liver and kidney lysosomes.

Lysosomes were isolated from the livers and from the kidneys of rats treated or not treated with the cysteine proteinase inhibitor leupeptin, and the levels of the intralysosomal serum albumin of the leupeptin-treated rats were compared with those of the saline-treated control rats. Leupeptin caused an intralysosomal accumulation of albumin in vivo because of its potent inhibition of lysosomal protein degradation. In fact, the lysosomes isolated from the livers and kidneys of leupeptin-treated rats almost completely lost their ability to degrade rat albumin in vitro. These findings show that the lysosomes are subcellular sites of the degradation of unlabeled serum albumin in these tissues. They also suggest that cysteine proteinases sensitive to leupeptin are involved in the lysosomal degradation of albumin. Albumin was degraded by total lysosomal enzymes in vitro. It was also degraded by the lysosomal extract being devoid of cathepsins H and J, prepared from rat kidney. The degradation of albumin by total lysosomal enzymes in vitro was greatly suppressed by a cysteine proteinase inhibitor, cystatin alpha, with no inhibition of cathepsins B and L. It was slightly suppressed by N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prol ine (CA-074), a selective inhibitor of cathepsin B, and by pepstatin, an inhibitor of cathepsin D, whereas it was markedly suppressed by a combination of cystatin alpha and either CA-074 or pepstatin. These and associated findings show that cystatin alpha-sensitive cysteine proteinase(s), which is distinct from cathepsins B, H, L, and J, and cathepsins B and D are involved in the lysosomal degradation of albumin.

Molecular weight distribution of hydrolysis products during biodegradation of model macromolecules in suspended and biofilm cultures I. Bovine serum albumin

Macromolecules can comprise a significant portion of dissolved organic carbon in wastewater and affect wastewater treatability by engineered systems, but little information is available about mechanisms of macromolecule degradation. The release of protein hydrolytic fragments was monitored during the degradation of the model protein bovine serum albumin (BSA) in batch and continuous suspended cultures and in fixed-film reactor systems. Three different inocula that represent a range of bacterial diversity were used: a protein degrading isolate, a commercial biological oxygen demand (BOD) test inoculum (limited diversity culture) and a wastewater inoculum. Molecular size distributions were monitored during degradation using membrane ultrafiltration techniques. Intermediate-molecular-weight protein hydrolytic fragments (2000–10,000 amu) were produced and released into solution by all cultures in all reactor types investigated. In batch suspended culture reactors with initial BSA concentrations of 100 mg liter −1, maximum concentrations of intermediate-molecular-weight hydrolytic fragments of 25, 10 and 6 mg liter −1 were found, respectively, in pure, limited diversity and wastewater cultures. Since material in the 2000–10,000 amu fraction decreased as culture diversity increased, it was concluded that the involvement of many microbial species during protein degradation limits the accumulation of hydrolytic fragments under conditions typical of wastewater treatment systems. These results lead to the proposal of a protein degradation mechanism that features cell-bound hydrolysis of protein and the subsequent release of hydrolytic fragments back into bulk solution. Hydrolysis and release is repeated by the same or different cells until fragments are small enough (< 1000 amu) to be directly assimilated by cells. Because the concentration of protein hydrolysis fragments released in high-diversity wastewater inoculated reactors was low and they were rapidly degraded, the release of hydrolysis fragments should have little impact on overall macromolecule degradation kinetics in domestic wastewater treatment systems.

Fluorometric analysis of endocytosis and lysosomal proteolysis in the rat visceral yolk sac during whole embryo culture.

Using spectrofluorimetry and fluorescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) during whole embryo culture. Rat conceptuses exposed continuously to FITC-albumin had linear increases of both acid-soluble and acid-insoluble FITC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumulated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of fluorescence. During a chase period following a pulse exposure to FITC albumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of proteinase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluorescence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluorescence microscopy of yolk sacs exposed to FITC-albumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and decreased substantially during a chase period. In conceptuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC-fluorescence whose clearance in pulse-chase experiments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125l-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of the effects of embryotoxicants on yolk sac function during whole embryo culture.

Albumin and hemalbumin degradation by Porphyromonas gingivalis.

Degradation of bovine albumin and hemalbumin by Porphyromonas gingivalis W50 cells under non-reducing conditions at 37 degrees C was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Albumin and hemalbumins with heme:protein molar ratios of 1:1, 4:1 and 8:1 were degraded, yielding protease-resistant 55.6-kDa peptides. Cells of strains WPH 35, 11834 and Bg 381 also produced a similar digestion pattern. N-terminal sequencing of the 55.6-kDa albumin digestion fragment revealed two peptides with the sequences 82glu-thr-tyr-gly-asp-met-ala and 95gln-pro-glu-arg-asn-glu-cys, indicating cleavage in the N-terminal hinge region. Tosyllysylchloromethylketone and N-ethylmaleimide were the most effective in inhibiting breakdown of albumin and hemalbumin with a 1:1 heme:protein ratio. Initial degradation rates of albumin and all hemalbumins were similar, but the total amount of hemalbumins degraded over 7.5 h decreased with increased ratio of bound hemin. The specific proteolysis of hemalbumin may enable P. gingivalis to release hemin from a region of the molecule where heme binding is least avid.

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

Degradation of a protein via the ubiquitin system involves two discrete steps, signaling by covalent conjugation of multiple moieties of ubiquitin and degradation of the tagged substrate. Conjugation is catalyzed via a three-step mechanism that involves three distinct enzymes that act successively: E1, E2, and E3. The first two enzymes catalyze activation of ubiquitin and transfer of the activated moiety to E3, respectively. E3, to which the substrate is specifically bound, catalyzes formation of a polyubiquitin chain that is anchored to the targeted protein. The polyubiquitin-tagged protein is degraded by the 26 S proteasome, and free and reutilizable ubiquitin is released. In addition to the three conjugating enzymes, targeting of certain proteins requires association with ancillary proteins and/or post-translational modification(s). Using a specific antibody to deplete cell extract from the molecular chaperone Hsc70, we demonstrate that this protein is required for the degradation of actin, alpha-crystallin, glyceraldehyde-3-phosphate dehydrogenase, alpha-lactalbumin, and histone H2A. In contrast, the degradation of bovine serum albumin, lysozyme, and oxidized RNase A is Hsc70-independent. Mechanistic analysis revealed that the chaperone is required for the conjugation reaction; however, it does not substitute for E3. Involvement of the chaperone in the proteolytic process requires complex formation with the substrate. Formation of this complex appears to be essential in the proteolytic process. In addition, the proper function of the chaperone in the proteolytic process requires the presence of K+, which allows rapid cycles of dissociation and association of the complex. The chaperone may act by binding to the substrate and unfolding it to expose a ubiquitin ligase-binding site. In addition, it can also act directly on the ubiquitination machinery.

Methylglyoxal-modified arginine residues — a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive α-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and N δ -(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG min-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4°C: the binding constant K d value was 377±35 nM and the number of receptors per cell was 5.9±0.2×10 5 ( n=12). N α -Acetyl- N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine displaced MG min-HSA from THP-1 cells, suggesting that the N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37°C, MG min-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG min-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.

Hydrogen peroxide-mediated degradation of protein: different oxidation modes of copper- and iron-dependent hydroxyl radicals on the degradation of albumin

Cupric ions (Cu 2+) added to hydrogen peroxide (H 2O 2) were found to generate hydroxyl radicals (HO ·) capable of benzoate hydroxylation. Although ferrous (Fe 2+) and ferric (Fe 3+) ions, when added to H 2O 2, resulted in very little production of HO ·, the addition of EDTA to the reaction mixture markedly increased their catalytic activity. In the absence of albumin, catalase (a H 2O 2 scavenger) and mannitol (an HO · radical scavenger) effectively inhibited the formation of HO · in H 2O 2/Cu 2+ and H 2O 2/Fe 2+/EDTA oxidation systems. On analysis using SDS-polyacrylamide gel electrophoresis, catalase was shown to prevent the degradation of albumin by both oxidation systems, whereas mannitol was an effective scavenger of the H 2O 2/Fe 2+/EDTA oxidation system but not of the H 2O 2/Cu 2+ oxidation system. Furthermore, the effect of alteration of benzoate hydroxylation and H 2O 2 consumption on the H 2O 2/Cu 2+ and H 2O 2/Fe 2+/EDTA oxidation systems resulted in opposite behavior that was dependent upon the presence or absence of albumin. These observations suggest that copper ions bind to albumin and induce site-specific degradation by HO · generated at the copper-binding site, whereas the Fe 2+/EDTA-catalyzed oxidation system induces non-specific degradation of albumin by HO · generated by the Fenton reaction between H 2O 2 and free Fe 2+/EDTA in solution.

Accumulation of Pyrraline-modified Albumin in Phagocytes due to Reduced Degradation by Lysosomal Enzymes

Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes.

Crystal growth in dental enamel: the role of amelogenins and albumin.

Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.