High Molecular Mass Aggregates Research Articles

Biochemical Characterization of the Small Heat Shock Protein IbpB from Escherichia coli

Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomal ibp operon and purified by refolding. Under our experimental conditions, IbpB exhibited pronounced size heterogeneity. Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100-200-nm range, which themselves associated to yield loose aggregates of micrometer size. IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly beta-pleated secondary structure. Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into approximately 600-kDa oligomers, and an increase in surface hydrophobicity. Structural changes were reversible between 37 and 55 degrees C, and, up to 55 degrees C, hydrophobic sites were reburied upon cooling. IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition. However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features. By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures. Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB.

The oligomeric state of Bacillus thuringiensis Cry toxins in solution

The molecular mass of different Cry toxins produced by Bacillus thuringiensis bacteria was estimated by size-exclusion chromatography and non-denaturing polyacrylamide gel electrophoresis at neutral and alkaline pH in order to assess the existence of oligomers in solution. We found that Cry1Aa, Cry1Ac, Cry1C, Cry1D and Cry3A toxins exist in solution as a mixture of monomer and high molecular mass aggregates with an apparent molecular mass greater than 600 kDa, that depend on the time elapsed between toxin activation and analysis. Aggregation of toxins by disulfide bonds is unlikely because aggregates are also observed in samples incubated with DTT. These data show that the Cry toxins studied do not form oligomers of less than ten subunits in solution and suggest that oligomer formation may occur after the toxin binds to the receptor and inserts into the membrane.

Electrophoretic characterization of the protein products formed during heat treatment of whey protein concentrate solutions

Whey protein concentrate (WPC) solutions containing 10, 30, 60 and 120 g dry powder/kg were heated at 75°C and whey protein aggregation was studied by following the changes in the distribution of β-lactoglobulin, α-lactalbumin and bovine serum albumin, using one dimensional and two dimensional PAGE. The one dimensional PAGE results showed that a minimal quantity of large aggregates was formed when 10 g WPC/kg solutions were heated at 75°C for up to 16 min whereas appreciable quantities were formed when 30, 60 and 120 g WPC/kg solutions were similarly treated. The two dimensional PAGE analysis showed that some disulphide-linked β-lactoglobulin dimers were present in heated 10 g WPC/kg solution, but very little was present in heated 120 g WPC/kg solution. By contrast, SDS was able to dissociate monomeric protein from high molecular mass aggregates in heated WPC solution of 120 g/kg but not in 10 g WPC/kg solution heated for 30 min. The rates of loss of native-like and SDS-monomeric β-lactoglobulin, α-lactalbumin and bovine serum albumin during heating increased as the WPC concentration was increased from 10 to 120 g/kg. In 120 g WPC/kg solution heated at 75°C, the amounts of SDS-monomeric β-lactoglobulin in each sample were greater than the quantities of native-like protein. However, in WPC solutions of 10, 30 and 60 g/kg, the differences between the amounts of native-like and SDS-monomeric proteins were slight. The loss of the native-like or SDS-monomeric proteins was consistent with a first or second order reaction. In each case, the apparent reaction rate constant appeared to be concentration-dependent, suggesting a change of aggregation mechanism in the more concentrated solutions. Overall, these results indicate that in addition to disulphide-linked aggregates, hydrophobic aggregates involving β-lactoglobulin, α-lactalbumin and bovine serum albumin were formed in heated WPC solution at high protein concentration, as suggested by model studies using binary mixtures of these proteins.

The junction-resolving enzyme T7 endonuclease I: quaternary structure and interaction with DNA

Endonuclease I is a DNA junction-selective resolving enzyme from bacteriophage T7. Using a nuclease-defective mutant that retains normal binding to DNA we show that the protein binds to four-way DNA junctions as a dimer, in common with other junction-resolving enzymes studied. Gel filtration and chemical crosslinking indicate that endonuclease I also exists in free solution as a dimer together with a tetramer and higher molecular mass aggregates. However, in marked contrast with other junction-resolving enzymes, there is no detectable subunit exchange under normal conditions. Only by exposure to 6 M urea could we induce subunit exchange, and this was used to generate heterodimeric species containing one active and one inactive subunit. Using a supercoil-stabilised cruciform substrate we demonstrate that an active subunit of endonuclease I can act as a junction-specific nuclease in a heterodimeric combination with an inactive subunit. However, the two subunits of a fully active homodimeric enzyme each cleave the phosphodiester backbone of a cruciform within the lifetime of the DNA-protein complex.

Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin.

alpha A-crystallin (alpha A) and alpha B-crystallin (alpha B) are among the predominant proteins of the vertebrate eye lens. In vitro, the alpha-crystallins, which are isolated together as a high molecular mass aggregate, exhibit a number of properties, the most interesting of which is their ability to function as molecular chaperones for other proteins. Here we begin to examine the in vivo functions of alpha-crystallin by generating mice with a targeted disruption of the alpha A gene. Mice that are homozygous for the disrupted allele produce no detectable alpha A in their lenses, based on protein gel electrophoresis and immunoblot analysis. Initially, the alpha A-deficient lenses appear structurally normal, but they are smaller than the lenses of wild-type littermates. alpha A-/- lenses develop an opacification that starts in the nucleus and progresses to a general opacification with age. Light and transmission electron microscopy reveal the presence of dense inclusion bodies in the central lens fiber cells. The inclusions react strongly with antibodies to alpha B but not significantly with antibodies to beta- or gamma-crystallins. In addition, immunoblot analyses demonstrate that a significant portion of the alpha B in alpha A-/- lenses shifts into the insoluble fraction. These studies suggest that alpha A is essential for maintaining lens transparency, possibly by ensuring that alpha B or proteins closely associated with this small heat shock protein remain soluble.

Chemical cross-linking leads to two high molecular mass aggregates of rat α1β1 integrin differing in their conformation but not in their composition

In order to detect protein interactions of the collagen/laminin receptor α 1 β 1 integrin, covalent chemical cross-linking was performed with the homo-bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross-linking of the 190 kDa rat α 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes ( M r 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of α 1 and β 1 integrin subunits. No other cross-linked proteins associated with the α 1 or β 1 subunit were detected. In contrast to the non-cross-linkable α 1 β 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of α 1 β 1 integrin to cross-link and produce two additional high molecular mass forms is shared by rat α 9 β 1 integrin. Thus, the cross-linking approach directly indicates that β 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.

In vitro cross-linking of calf lens alpha-crystallin by malondialdehyde.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.

Enhancement of Pasteurella haemolytica leukotoxic activity by bovine serum albumin

Summary Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (bsa) for 2.5 hours enhanced culture supernatant leukotoxic activity (30,700 ± 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 ± 40 toxic units/ml). Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + bsa) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + bsa to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that Phaemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of bsa, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Transglutaminases catalyze cross-linking of plasminogen to fibronectin and human endothelial cells.

We have previously reported that apolipoprotein (a) is a substrate for transglutaminases. We now demonstrate that plasminogen which is homologous to apolipoprotein (a), is also modified by these enzymes. Transglutaminases from different sources mediated the incorporation of monodansyl-cadaverine into plasminogen, indicating the presence of reactive glutamine(s) in plasminogen. Reactive lysines were also identified using the lysine-decorating peptide dansyl-PGGQQIV. In addition, transglutaminases catalyzed the formation of plasminogen homopolymers and plasminogen-fibronectin heteropolymers. Human umbilical vein endothelial cells cross-linked plasminogen into high molecular mass aggregates. Cross-linked plasminogen was cell associated, and no cross-linking of plasminogen was seen in the fluid-phase. Large molecular mass plasminogen generated on the human umbilical vein endothelial cell (HUVEC) surface could not be eluted with epsilon-aminocapoic acid and was activatable by tissue plasminogen activator. These results suggest that, following non-covalent association of plasminogen with the HUVEC surface, cell surface-associated transglutaminase catalyzes cross-linking of plasminogen into large molecular mass aggregates that can be converted into functional plasmin. It is proposed that transglutaminases may function to localize plasminogen to cell surfaces and matrices of tissues.

Competitive inhibition of lipolytic enzymes VIII: Inhibitor-induced aggregation of porcine pancreatic phospholipase A 2

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was observed only when incorporated into a micellar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.

Sulfur starvation in Lemna leads to degradation of ribulose-bisphosphate carboxylase without plant death.

Little is known about the degradation of the most abundant protein in nature, ribulose-bisphosphate carboxylase (RuBP carboxylase, EC 4.1.1.39), probably reflecting the fact that no stress situation has been identified capable of causing extensive RuBP carboxylase degradation without causing the death of the plant. We have subjected plants of Lemna minor L. to a variety of stress situations, nutritive deficiencies in particular, and have found a single condition--sulfur starvation--that caused almost complete degradation of RuBP carboxylase without causing plant death. Moreover, the enzyme was preferentially degraded under these conditions. However, when the plants were deprived of calcium, no RuBP carboxylase degradation was observed. Instead, the enzyme was oxidized and polymerized into high molecular mass aggregates. On the other hand, RuBP carboxylase shows an extreme stability when Lemna is deprived of some macronutrients (e.g. nitrogen, phosphorus, potassium, and magnesium) probably reflecting that this plant had to evolve in a way to cope with frequent shortages of such elements. The implications of these data for the role of RuBP carboxylase as a leaf storage protein are discussed.

N-acetyl- p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes

Incubation of isolated rat hepatocytes with N-acetyl- p-benzoquinone imine (NAPQI) or 3,5-dimethyl- N-acetyl- p-benzoquinone imine (3,5-Me 2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me 2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me 2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me 2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPOI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.

Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase

5'-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5'-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5'-nucleotidase showed "break points" for all detergents in the temperature range 30-37 degrees C. SDS-PAGE of pure 5'-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200-630 kDa, suggesting that lymphocyte 5'-nucleotidase may be present in high molecular mass aggregates in its native state.

Carbonate dehydratase (carbonic anhydrase) in a spider. Association with the hemolymph lipoprotein.

Carbonate dehydratase was detected dissolved in the hemolymph of the tarantula, Eurypelma californicum. The enzyme was purified 31-fold by gel filtration, anion-exchange chromatography, a second gel filtration, and finally, preparative polyacrylamide gel electrophoresis. Zinc content increased during purification to up to 2.4 mol Zn/100 000 g of protein (= 1.58 mg Zn/g protein). In the polyacrylamide electrophoresis of tarantula hemolymph under non-denaturing conditions three major protein bands were observed: hemocyanin, a 16 S lipoprotein and the active band which migrated closely behind the 16 S lipoprotein. After treatment with sodium dodecyl sulfate both the carbonate dehydratase-active protein and the lipoprotein revealed bands corresponding to Mr = 95 000 and 110 000, respectively, but the enzymatically active protein revealed an additional third band with Mr = 40 000. The latter band is though to represent the 'true' carbonate dehydratase protein. Upon isoelectric focusing of material containing carbonate dehydratase activity and lipoprotein, bands were obtained at pH 5.45, 5.6 and 5.7. The band at pH 5.6 contained the peak of enzyme activity, and upon dodecyl sulfate-polyacrylamide gel electrophoresis showed the highest proportion of the 40-kDa polypeptide. It is concluded that tarantula carbonate dehydratase, instead of forming a high molecular mass aggregate, is associated with the 16 S lipoprotein, the latter serving as a carrier for the enzyme. The lipoprotein is probably also involved in other transport processes. It is present in great excess and may therefore occur in two forms, charged with carbonate dehydratase or uncharged. Tarantula carbonate dehydratase is inhibited by acetazolamide and by dansylamide, but not by a number of other known inhibitors, most notably not by 4-(aminomethyl)benzenesulfonamide. Treatment with 1M urea does not affect specific enzyme activity, while 2M urea inhibits by 50%. 2-Mercaptoethanol inhibits activity by 50% at 0.1M. Like other carbonate dehydratases, the tarantula enzyme shows esterase activity. The Km for 4-nitrophenyl acetate is 5mM.