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PubMed2005

The recognition of adsorbed and denatured proteins of different topographies by beta2 integrins and effects on leukocyte adhesion and activation.

Leukocyte beta2 integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography of a protein adlayer. Albumin adsorbed from the native conformation gave rise to different adlayer topographies and different amounts of adsorbed protein on hydrophobic and relatively hydrophilic polystyrene and silanised silicon-wafer surfaces, whereas adsorption of pre-denatured Alb resulted in similar adlayer topographies and similar amounts of adsorbed protein on these surfaces. All three distinct protein-adlayer topographies supported adhesion of in vitro differentiated, macrophage-like U937 and THP-1 cells, but did not support adhesion of their promonocytic precursors. Human monocytes freshly isolated from peripheral blood did not adhere to adsorbed albumin, not even in the presence of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1alpha chemokines. Adhesion of the macrophage-like cells to albumin in any of the three topographies was inhibited by antibodies against beta2 integrins, but not by antibodies against beta1 integrins, and did not induce secretion of the proinflammatory cytokine tumour necrosis factor-alpha.

Adsorption↗
PubMed1989

Macroxyproteinase (M.O.P.): a 670 kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells.

Erythrocytes and reticulocytes are shown to undergo rapid rates of protein degradation following exposure to oxidative stress. Experiments with ATP depletion revealed that, unlike the proteolysis of many other abnormal proteins, the degradation of oxidatively modified proteins is an ATP-independent process. Ion exchange chromatography (DEAE Sepharose CL-6B), ammonium sulfate precipitation, gel filtration chromatography (Sephacryl S-300 or Sepharose CL-6B), and a second ion exchange step were used to resolve the activity responsible for degrading oxidatively modified proteins from (dialyzed) cell-free extracts of erythrocytes and reticulocytes. Gel filtration studies revealed that some 70-80% of the activity in erythrocytes, and some 60-70% of the activity in reticulocytes, is expressed by a 670 kDa proteinase complex that is not stimulated by ATP (in fact, ATP is slightly inhibitory). This proteinase complex is inhibited by sulfhydryl reagents, serine reagents, and transition metal chelators, and has a pH optimum of 7.8. We propose the trivial name "macroxyproteinase" or "M.O.P." (abbreviated from Macro-Oxy-Proteinase) for the complex because of its large size, substrate preference (oxidatively modified proteins), and inhibitor profile (which indicates multiple catalytic sites). Electrophoresis studies of the 670 kDa M.O.P. complex revealed the presence of 8 distinct polypeptide subunits with the following apparent molecular sizes: 21.5, 25.3, 26.2, 28.1, 30.0, 31.9, 33.3, and 35.7 kDa. The large molecular size of the M.O.P. complex, its ATP- and ubiquitin-independence, its inhibitor profile, its distinctive subunit banding pattern in denaturing electrophoresis gels, its pH optimum, and its proteolytic profile with fluorogenic peptide substrates all indicate that M.O.P. is identical to 600-700 kDa neutral/alkaline proteinase complexes that have been isolated from a wide variety of eucaryotic cells and tissues, but for which no function has previously been clear. We propose that macroxyproteinase is responsible for catalyzing most of the selective degradation of oxidatively denatured proteins in red blood cells. We further suggest that M.O.P. may perform the same function in other eucaryotic cells and tissues.

Adult↗
PubMed2005

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration.

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.

Animals↗
PubMed1977

Structural analysis of denaturant-protein interactions: comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme.

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.

Ethanol↗
PubMed2001

Protein denaturation described by a two-state structural model of liquid water.

Our laboratory has been involved in expounding an outer neighbor molecular-level structural theory for liquid water in the supercooled and ordinary thermodynamic regimes. This theory, which depends on transformations with increasing temperature or pressure, is consistent with all the properties of this substance, including the ten or so "anomalies", and has been recently used to explain the effect that surrounding water has on proteins. For example, the sharp decrease in the hydration entropies of polar groups can be explained through a consideration of the promotion of ice-Ih-type bonding structure at the expense of the less stable ice-II-type bonding structure. These structural transformations occur in the local neighborhood of the polar group. In this paper we discuss this outer neighbor two-state structural theory for liquid water, the role it plays in explaining water's anomalous properties and its description of protein denaturation both as a function of temperature and pressure.

Mathematics↗
PubMed1981

Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments.

The complete denaturation and subsequent renaturation and reconstitution of a polytopic integral membrane protein are demonstrated. Delipidated bacteriorhodopsin (Huang, K.-S., Bayley, H., and Khorana, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 323-327) is completely denatured when transferred into 88% formic acid or anhydrous trifluoroacetic acid as shown by NMR and circular dichroism spectroscopy. When ethanol is added to a solution of the denatured protein, helical structure is largely reformed. After neutralization of the acid with ammonia and dialysis against a solution of sodium dodecyl sulfate a substantial amount of this structure is retained. Complete renaturation, characterized by the formation of the chromophore, occurs when phospholipids, cholate, and retinal are added to the sodium dodecyl sulfate solution of the protein. After dialysis of the solution to remove the detergents, the bacteriorhodopsin assembles into vesicles that are fully active in light-driven proton translocation. We also show that two chymotryptic fragments of bacteriorhodopsin (residues 1-71 and 72-248), separated under denaturing conditions, can be made to reassociate and form active vesicles with phospholipids.

Apoproteins↗
PubMed1989

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

Animals↗
PubMed1993

Attempts to restore scrapie prion infectivity after exposure to protein denaturants.

A wealth of experimental evidence argues that infectious prions are composed largely, if not entirely, of the scrapie isoform of the prion protein. We attempted to restore scrapie infectivity after exposure to protein denaturants including urea, chaotropic salts, and SDS. None of the procedures restored infectivity. Dialysis to remove slowly chaotropic ions and urea failed to restore scrapie infectivity. Attempts to create monomers of the scrapie isoform of the prion protein under nondenaturing conditions using a wide variety of detergents have been unsuccessful, to date, except for one report claiming that scrapie infectivity could be recovered from 12% polyacrylamide gels after SDS/PAGE [Brown, P., Liberski, P. P., Wolff, A. & Gajdusek, D. C. (1990) Proc. Natl. Acad. Sci. USA 87, 7240-7244]. We found that < 0.001% of the infectious prion titer could be recovered from the region of a polyacrylamide gel where the denatured proteinase K-resistant core of the scrapie isoform of the prion protein and other 30-kDa proteins migrate. We conclude that under the denaturing conditions used for SDS/PAGE, the scrapie isoform of the prion protein is denatured and little or no renaturation occurs upon injection of fractions eluted from gels into animals for bioassays.

Animals↗
PubMed1975

[Protein denaturation in frozen fish].

To inhibit the rapid deterioration of the production-technological properties of fish meat minced and frozen on board, it is necessary to become acquainted with the causes of protein denaturation under these conditions. It was found that the protein solubility in codfish is impaired by formaldehyde which develops from trimethyl-amine oxide during storage and also by the salt content. After 7 days at -20 degrees C, the solubility of the sarcoplasmic and myofibrillary proteins in minced fish meat added with 80 mg of formaldehyde per 100 g of total protein amounted to almost 70% and 35%, respectively, of the value determined in controls, i.e., samples without formaldehyde. After this period of storage, only 30% of the added formaldehyde were in the free state. After 1 month, the solubility of the myofibrillary proteins in waterleached fish meat was by 30% higher than in unleached controls, 80 mg of formaldehyde per 100 mg of total protein having been added in both cases. After 1 month at -5 degrees C and -20 degrees C, only 90 and 15 p.p.m. of formaldehyde, respectively, were found in minced fish meat, whereas leached samples contained no formaldehyde. The solubility of the myofibrillary proteins in leached fish meat (the initial salt content of which had been restituted) was by 30% lower than in the unleached controls.

Animals↗
PubMed2001

Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native protein in the dried state as well as the complete recovery of active beta-gal on reconstitution. These results indicate that addition of an effective stabilizer, such as sucrose, may minimize protein denaturation during freeze-drying in phosphate buffers, even if there are large-scale changes in solution pH during freezing.

Buffers↗
PubMed1993

Denatured proteins inhibit translation in hemin-supplemented rabbit reticulocyte lysate by inducing the activation of the heme-regulated eIF-2 alpha kinase.

The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initiation factor eIF-2. The molecular nature of the "signal" that leads to the activation of HRI in response to heat shock has not been characterized. We have recently reported that HRI interacts with the 90- and 70-kDa heat shock proteins (hsp) and a 56-kDa protein in hemin-supplemented lysates [Matts, R.L., Xu, Z., Pal, J.K., & Chen, J.-J. (1992) J. Biol. Chem. 267m 18160-18167]. In this report, we demonstrate that addition of denatured proteins, bovine serum albumin (BSA), beta-lactoglobulin, or alpha-lactalbumin, but not the addition of the native proteins, inhibits protein synthesis in hemin-supplemented reticulocyte lysates. The inhibition was reversed upon the addition of 10 mM cAMP or purified eIF-2B, classical criteria for HRI-mediated translational inhibition. Denatured BSA, but not native BSA, stimulated the phosphorylation of the alpha-subunit of eIF-2. This stimulation of eIF-2 alpha phosphorylation was inhibited by a monoclonal antibody to HRI, confirming that denatured BSA was causing the activation of HRI. The concentration of denatured BSA required to inhibit protein synthesis by 50% correlated with the levels of hsp70 present in each lysate preparation. Lysate hsp70 co-immunoadsorbed with denatured BSA, but not with not with native BSA. Hsp70 was co-adsorbed with HRI from lysate in the presence of native BSA, but not in the presence of denatured BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Analysis of protein denaturation by high-performance continuous differential viscometry.

The intrinsic viscosity of protein solutions changes when denaturation occurs. Therefore, viscosity measurement using traditional glass capillary viscometers has been a key method to study the process of protein denaturation. Such measurements are laborious, time consuming and need at least 10 ml of sample. The Viscotek differential viscometer can be used as an on-line detector for high-performance liquid chromatography to monitor the viscosity of column effluent. In this study, samples were injected using an autosampler onto a "delay" column containing glass beads in place of the high-performance liquid chromatography columns. Results indicate guanidine hydrochloride, heat and pH act as denaturing agents and changes the intrinsic viscosities of serum albumin, turkey egg albumin, and ovalbumin solutions. The differential viscometer is sensitive and provides accurate measurements of minor changes in viscosities of very dilute protein solutions undergoing denaturation. The advantage of using the differential viscometer instead of conventional glass capillary viscometer is the increased sensitivity, precision, speed and operational ease that permits measurements of solution viscosity of low sample concentrations up to 1.2 micrograms of pure proteins.

Animals↗
PubMed1994

NMR, alcohols, protein solvation and protein denaturation.

The amino acid side chains of Ser, Thr and Tyr contain the hydroxyl functional group that is characteristic of alcohols, and a variety of different alcohols are used either as structure-inducing or denaturing co-solvents in physical-chemical studies of proteins. This article surveys selected aspects of the influence of protein-intrinsic and -extrinsic hydroxyl groups on studies of the solvation of peptides and proteins by nuclear magnetic resonance (NMR) spectroscopy.

Alcohols↗
PubMed2005

Interaction of a small heat shock protein of the fission yeast, Schizosaccharomyces pombe, with a denatured protein at elevated temperature.

We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 degrees C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 degrees C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 degrees C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 degrees C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.

Biochemistry↗
PubMed1975

A method for the identification and quantitative investigation of denatured proteins in mixtures based on computer comparison of amino-acid patterns.

A procedure is described in which stepwise regression is adapted to permit comparison of the amino-acid pattern from a composite sample with those of simple substances arranged in an easily accessible data bank. The computer program automatically selects from the bank those proteins whose amino-acid patterns best correspond to that of the sample, and calculates the proportion of the proteins contained in the mixture. The procedure is applicable to food analytical problems that involve the identification and determination of proteins in mixtures, and also to situations in which the properties of the proteins have been altered by denaturation or enzymatic degradation. The determination is limited to 3-4 proteins in the mixture.

Amino Acids↗
PubMed2000

Enhanced protein denaturation in indomethacin-treated cells.

Indomethacin, a potent anti-inflammatory drug, activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1), but this is insufficient to elevate heat shock gene expression. However, indomethacin pretreatment leads to a complete heat shock response at temperatures that are by themselves insufficient. Here, we showed that the heat-induced loss of enzymatic activity of a nuclear or a cytoplasmic luciferase expressed in murine cells was enhanced when cells had been pretreated with indomethacin. Additionally, in these cells the 70-kDa constitutive heat shock protein exhibited an enhanced aggregation in the presence of indomethacin. Similarly an increase in the aggregation of beta-galactosidase was observed. These data suggest that indomethacin at moderate temperatures accelerates the presence of denatured proteins in the cell, thus lowering the temperature threshold for a heat shock response.

3T3 Cells↗