Biomedical subjects

A Azem

Publications and source records attributed to A Azem.

18 recordsLinked to original sources

PubMed2001

The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60.

Mitochondrial chaperonins are necessary for the folding of newly imported and stress-denatured mitochondrial proteins. The goal of this study was to investigate the structure and function of the mammalian mitochondrial chaperonin system. We present evidence that the 60 kDa chaperonin (mt-cpn60) exists in solution in dynamic equilibrium between monomers, heptameric single rings and double-ringed tetradecamers. In the presence of ATP and the 10 kDa cochaperonin (mt-cpn10), the formation of a double ring is favored. ADP at very high concentrations does not inhibit malate dehydrogenase refolding or ATP hydrolysis by mt-cpn60 in the presence of mt-cpn10. We propose that the cis (mt-cpn60)14.nucleotide.(mt-cpn10)7 complex is not a stable species and does not bind ADP effectively at its trans binding site.

Adenosine Diphosphate↗

PubMed2001

Comparison of body fluid distribution between chronic haemodialysis and peritoneal dialysis patients as assessed by biophysical and biochemical methods.

BACKGROUND: The control of extracellular volume is a key parameter for reducing hypertension and the incidence of cardiovascular mortality in dialysis patients. In recent years bioimpedance measurement (BIA) has been proven as a non-invasive and accurate method for measuring intracellular and extracellular fluid spaces in man. In addition, plasma atrial natriuretic peptide (ANP) and cyclic guanosine monophosphatase (cGMP) concentrations have been shown to reflect central venous filling. Using these methods, we compared body fluid status between stable patients on haemodialysis and peritoneal dialysis. METHODS: Thirty-nine chronic haemodialysis patients, 43 chronic peritoneal dialysis patients and 22 healthy controls were included in the study. Multifrequency BIA was performed using the Xitron BIS4000B device (frequencies from 5 to 500 kHz were scanned and fitted) in patients before and after haemodialysis. Peritoneal dialysis patients were measured after drainage of the dialysate. Plasma ANP and cGMP levels were measured in plasma using a (125)I solid phase RIA. Serum albumin concentrations and serum osmolality were measured in all patients. The body fluid data were analysed in relation with the clinical findings. RESULTS: Total body water (TBW) was 0.471+/-0.066 l/kg before haemodialysis and 0.466+/-0.054 l/kg after haemodialysis. Peritoneal dialysis patients had a TBW (0.498+/-0.063 l/kg) that was greater than the before and after dialysis values of haemodialysis patients. The extracellular body fluid (V(ecf)) was increased pre-haemodialysis. It was even greater in peritoneal dialysis patients compared with patients both pre- and post-haemodialysis (pre 0.276+/-0.037 l/kg; post 0.254+/-0.034 l/kg; peritoneal dialysis 0.293+/-0.042 l/kg, P<0.05). However, plasma ANP concentrations (representing intravascular filling) in peritoneal dialysis patients were comparable with post-haemodialysis values (284+/-191 pg/ml vs 286+/-144 pg/ml). The correlation coefficient between sysRR and V(ecf) was r=0.257 in haemodialysis (P=0.057) and r=0.258 in peritoneal dialysis (P<0.05). A significant negative correlation was found between serum albumin and V(ecf)/TBW in peritoneal dialysis patients (r= -0.624). CONCLUSION: Body fluid analysis by BIA demonstrated that TBW and V(ecf) were increased in peritoneal dialysis patients, and were comparable or even greater than values found before haemodialysis. However, plasma ANP levels indicated that intravascular filling was not increased in peritoneal dialysis. The ratio of V(ecf) to TBW was correlated to systolic pressure and negatively to serum albumin in peritoneal dialysis patients.

Adult↗

PubMed2000

Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding.

Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, alpha and beta. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the beta subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, alpha subunits only assemble in the presence of beta subunits. Although beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted alpha/beta 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

Adenosine Triphosphate↗

PubMed1999

Domain structure and lipid interaction of recombinant yeast Tim44.

Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region.

Cardiolipins↗

PubMed1998

Structural analysis of GroE chaperonin complexes using chemical cross-linking.

In this chapter, we have shown how chemical cross-linking with a bifunctional reagent, GA, can be used to investigate the structure of large oligomeric complexes such as GroEL14GroES7 and GroEL14(GroES7)2. Cross-linking, followed by denaturing electrophoresis, confirmed the number and arrangement of GroEL and GroES subunits within each individual oligomer, which was previously known from EM analysis. Furthermore, cross-linking permitted a close examination of the effect of regulatory factors, such as nucleotides and free divalent cations, on the molecular structure of GroEL14, GroEL14GroES7, and GroEL14GroES7. Finally, cross-linking analysis permitted characterization and quantitation of various chaperonin heterooligomeric complexes, GroEL14, GroEL14GroES7, and GroEL14GroES7 in solution, under conditions that also supported protein folding and ATP hydrolysis. It was shown that GA does not induce the artifactual association or the dissociation of GroES7 from the chaperonin. On the contrary, chemical cross-linking is an obligatory procedure when the subsequent analysis is carried out using methods that can displace the equilibrium.

Adenosine Triphosphate↗

PubMed1997

Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.

Bacterial Proteins↗

PubMed1997

The mitochondrial hsp70 chaperone system. Effect of adenine nucleotides, peptide substrate, and mGrpE on the oligomeric state of mhsp70.

Mitochondrial hsp70 (mhsp70) is a key component in the import and folding of mitochondrial proteins. In both processes, mhsp70 cooperates with the mitochondrial nucleotide exchange factor mGrpE (also termed Mge1p). In this work we have characterized the self-association of purified mhsp70, the interaction of mhsp70 with isolated mGrpE and protein substrate, and the effect of nucleotides on these interactions. mhsp70 can form oligomers that are dissociated by ATP or by a nonhydrolyzable ATP analog. A substrate peptide binds to mhsp70 in the absence of added nucleotides and is released by ATP but not by ADP. Binding of the peptide causes nucleotide-independent dissociation of the mhsp70 oligomers and enhances the mhsp70 ATPase. Purified mGrpE forms a homodimer. In the absence of added nucleotides, one mGrpE dimer binds to one molecule of mhsp70, forming a stable 122 kDa hetero-oligomer. This complex is weakened by ADP and completely dissociated by ATP.

Adenosine Diphosphate↗

PubMed1997

GroES binding regulates GroEL chaperonin activity under heat shock.

Chaperonins GroEL14 and GroES7 are heat-shock proteins implicated in the molecular response to stress. Protein fluorescence, crosslinking and kinetic analysis revealed that the bond between the two otherwise thermoresistant oligomers is regulated by temperature. As temperature increased, the affinity of GroES7 and the release of bound proteins from the chaperonin concomitantly decreased. After heat shock, GroES7 rebinding to GroEL14 and GroEL14GroES7 particles correlated with the restoration of optimal protein folding/release activity. Chaperonins thus behave as a molecular thermometer which can inhibit the release of aggregation-prone proteins during heat shock and restore protein folding and release after heat shock.

Adenosine Triphosphatases↗

PubMed1997

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Adenosine Diphosphate↗

PubMed1997

What is the driving force for protein import into mitochondria?

Nuclear-encoded mitochondrial proteins are synthesized in the cytosol as precursors and then imported into mitochondria. Protein import into the matrix space requires the function of the mitochondrial hsp70 (mhsp70) chaperone. mhsp70 is an ATPase that acts in conjunction with two partner proteins: the Tim44 subunit of the inner membrane import complex, and the nucleotide exchange factor mGrpE. A central question concerns how mhsp70 uses the energy of ATP hydrolysis to transport precursor proteins into the matrix. Recent evidence suggests that mhsp70 is a mechanochemical enzyme that actively pulls precursors across the inner membrane.

Adenosine Triphosphate↗

PubMed1995

The protein-folding activity of chaperonins correlates with the symmetric GroEL14(GroES7)2 heterooligomer.

Chaperonins GroEL and GroES form, in the presence of ATP, two types of heterooligomers in solution: an asymmetric GroEL14GroES7 "bullet"-shaped particle and a symmetric GroEL14(GroES7)2 "football"-shaped particle. Under limiting concentrations of ATP or GroES, excess ADP, or in the presence of 5'-adenylyl imidodiphosphate, a correlation is seen between protein folding and the amount of symmetric GroEL14(GroES7)2 particles in a chaperonin solution, as detected by electron microscopy or by chemical crosslinking. Kinetic analysis suggests that protein folding is more efficient when carried out by a chaperonin solution populated with a majority of symmetric GroEL14(GroES7)2 particles than by a majority of asymmetric GroEL14GroES7 particles. The symmetric heterooligomer behaves as a highly efficient intermediate of the chaperonin protein folding cycle in vitro.

Adenosine Diphosphate↗

PubMed1995

Increased efficiency of GroE-assisted protein folding by manganese ions.

This study addresses the role of ATP-bound and free Mg2+ and Mn2+ ions in the activation and modulation of chaperonin-assisted refolding of urea-denatured malate dehydrogenase. As compared with Mg2+, Mn2+ ions caused a significant increase in the rate of GroE-assisted malate dehydrogenase refolding and, concomitantly, a decrease in the rate of ATP hydrolysis. Moreover, Mn2+ increases the affinity of GroES for GroEL, even in the presence of saturating amounts of Mg2+. Chemical cross-linking showed that lower concentrations of Mn-ATP as compared with Mg-ATP are needed to form both asymmetric GroEL14GroES7 and symmetric GroEL14(GroES7)2 particles. The manganese-dependent increase in the rate of protein folding concurred with a specific increase in the amount of symmetric GroEL14-(GroES7)2 particles detected in a chaperonin solution. Thus, Mn2+ is a cofactor that can markedly increase the efficiency of the chaperonin reaction in vitro. Mn2+ ions can serve as an important tool for analyzing the molecular mechanism and the structure of chaperonins.

Adenosine Triphosphate↗

PubMed1995

Cross-linking of porin with glutardialdehyde: a test for the adequacy of premises of cross-linking theory.

Exposure of porin from Escherichia coli to glutardialdehyde followed by SDS-gel electrophoresis in 3% polyacrylamide yielded three bands that were identified in order of decreasing mobility as monomers and two and three cross-linked polypeptide chains. The distribution of protein among the three species for different extents of reaction showed a remarkably good agreement with corresponding values predicted from cross linking theory for an oligomer composed of three identical subunits arranged according to a 3-fold rotation axis. Electrophoresis performed in 7.5% polyacrylamide yielded four bands that were assigned to polypeptide chain monomers, dimers, and two types of trimers carrying two and three intersubunit cross-links. Our findings provide evidence that the central premise of cross-linking theory, viz. that the intersubunit cross-links formed upon exposure of a protein to a bifunctional reagent be governed by the symmetry of the molecule, is valid. Careful interpretation of cross-linking experiments thus proves an effective method to assess the oligomeric structure of a protein and reveal the symmetry underlying the spatial arrangement of the subunits within the molecule.

Cross-Linking Reagents↗

PubMed1995

Effect of free and ATP-bound magnesium and manganese ions on the ATPase activity of chaperonin GroEL14.

Hydrolysis of ATP by the GroEL14 chaperonin oligomer is activated and modulated by Mg2+ or Mn2+ ions. Mg-ATP and Mn-ATP can serve as substrates of the reaction and bind in a positively cooperative manner to the same catalytic sites on GroEL14, with similar binding constants in the micromolar range. In addition, millimolar amounts of Mg2+ and Mn2+ ions can further activate the GroEL14-ATPase while interacting with low-affinity noncatalytic sites on the chaperonin. The extent of ATPase activation by Mn2+ is half of that by Mg2+ ions. When both Mg2+ and Mn2+ ions are present in the same reaction, Mn2+ behaves as a noncompetitive partial inhibitor of the Mg-dependent ATPase. This inhibition requires the presence of ADP in the catalytic site. The binding affinity of Mn-ADP to the site is significantly higher than that of Mg-ADP. A slower release of Mn-ADP from the catalytic site thus changes the rate-determining step of the GroEL14-ATPase cycle. In the cell, the concentrations of Mg2+ and Mn2+ ions are such that both divalent ions may modulate chaperonin activity.

Adenosine Triphosphatases↗

PubMed1994

Characterization of a functional GroEL14(GroES7)2 chaperonin hetero-oligomer.

Chaperonins GroEL and GroES form two types of hetero-oligomers in vitro that can mediate the folding of proteins. Chemical cross-linking and electron microscopy showed that in the presence of adenosine triphosphate (ATP), two GroES7 rings can successively bind a single GroEL14 core oligomer. The symmetric GroEL14(GroES7)2 chaperonin, whose central cavity appears obstructed by two GroES7 rings, can nonetheless stably bind and assist the ATP-dependent refolding of RuBisCO enzyme. Thus, unfolded proteins first bind and possibly fold on the external envelope of the chaperonin hetero-oligomer.

Adenosine Triphosphate↗

PubMed1994

Effect of divalent cations on the molecular structure of the GroEL oligomer.

Structural analysis, by chemical cross-linking with glutardialdehyde (GA), and by urea denaturation, was carried out for the chaperonin oligomer GroEL14 from Escherichia coli. The cross-linking reaction of GroEL14 presents two phases: a rapid intralayer cross-linking reaction, which first occurs between the monomers of individual GroEL7 heptameric rings, and a slow interlayer cross-linking reaction, which later occurs between the two stacked heptameric rings of the GroEL14 oligomer. The biphasic behavior of the cross-linking reaction indicates that the surfaces of contact between GroEL monomers within individual heptameric rings are more extensive than the surfaces of contact between the two GroEL7 rings of the oligomer. Millimolar amounts of the divalent cations Mg2+, Mn2+, Ca2+, or Zn2+, but not of monovalent ions, increase the velocity of both intra- and interlayer cross linking. Divalent cations increase the stability of the native GroEL14 oligomer in urea. In contrast, Mg2+ activates ATP hydrolysis by GroEL14, with an activation constant in the micromolar range, while Ca2+ does not significantly assist ATP hydrolysis. It is concluded that divalent cations affect the structure of GroEL14 in particular the contacts between monomers within the GroEL7 heptameric layers. The effect of divalent cations on the structure of the chaperonin molecule is quantitatively and qualitatively distinct from that of magnesium ions on the chaperonin ATPase activity.

Adenosine Triphosphatases↗

PubMed1971

[Abdominal tumor].

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Abdominal Neoplasms↗

PubMedDate not supplied

Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.

Animals↗