scholarly journals Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments

1989 ◽  
Vol 264 (3) ◽  
pp. 671-677 ◽  
Author(s):  
C Méjean ◽  
F Pons ◽  
Y Benyamin ◽  
C Roustan
Keyword(s):  
Creatine Kinase ◽  
Conformational Changes ◽  
Contact Area ◽  
Binding Sites ◽  
Arginine Kinase ◽  
Glycolytic Enzymes ◽  
Actin Monomer ◽  
Terminal Part ◽  
Specific Antibodies ◽  
Antigenic Epitopes

The topology of the interfaces between actin monomers in microfilaments and three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase) was investigated using several specific antibodies directed against precisely located sequences in actin. A major contact area for glyceraldehyde-3-phosphate dehydrogenase was characterized in a region near residue 103. This interaction altered, by long-range conformational changes, the reactivity of antigenic epitopes in the C-terminal part of actin. The interface between actin and aldolase appeared to involve a sequence around residue 299 in the C-terminal region of actin. The interaction of phosphofructokinase, in contrast, modified the reactivity of all antibodies tested. Finally, the phosphagen kinases arginine kinase and creatine kinase showed no interaction with the microfilament.

scholarly journals Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces

1987 ◽  
Vol 244 (3) ◽  
pp. 571-577 ◽  
Author(s):  
C Mejean ◽  
M Boyer ◽  
J P Labbé ◽  
L Marlier ◽  
Y Benyamin ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Contact Area ◽  
Terminal Part ◽  
Specific Antibodies ◽  
Constant Sequence ◽  
Close Proximity ◽  
Immunological Approach

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.

scholarly journals Ligand-Induced conformational changes in the lactose permease ofescherichia coli: Evidence for two binding sites

1994 ◽  
Vol 3 (12) ◽  
pp. 2294-2301 ◽  
Author(s):  
Jianhua Wu ◽  
Stathis Frillingos ◽  
John Voss ◽  
H. Ronald Kaback
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Ofescherichia Coli ◽  
Lactose Permease

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals On the dynamics of some small structural motifs in rRNA upon ligand binding

2008 ◽  
Vol 73 (1) ◽  
pp. 41-53
Author(s):  
Aleksandra Rakic ◽  
Petar Mitrasinovic
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Binding Sites ◽  
De Novo ◽  
Reference Structure ◽  
Base Pairs ◽  
Rna Sequences ◽  
Conformational Model ◽  
Thermodynamic Variables ◽  
Dynamics Simulations

The present study characterizes using molecular dynamics simulations the behavior of the GAA (1186-1188) hairpin triloops with their closing c-g base pairs in large ribonucleoligand complexes (PDB IDs: 1njn, 1nwy, 1jzx). The relative energies of the motifs in the complexes with respect to that in the reference structure (unbound form of rRNA; PDB ID: 1njp) display the trends that agree with those of the conformational parameters reported in a previous study1 utilizing the de novo pseudotorsional (?,?) approach. The RNA regions around the actual RNA-ligand contacts, which experience the most substantial conformational changes upon formation of the complexes were identified. The thermodynamic parameters, based on a two-state conformational model of RNA sequences containing 15, 21 and 27 nucleotides in the immediate vicinity of the particular binding sites, were evaluated. From a more structural standpoint, the strain of a triloop, being far from the specific contacts and interacting primarily with other parts of the ribosome, was established as a structural feature which conforms to the trend of the average values of the thermodynamic variables corresponding to the three motifs defined by the 15-, 21- and 27-nucleotide sequences. From a more functional standpoint, RNA-ligand recognition is suggested to be presumably dictated by the types of ligands in the complexes.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

scholarly journals Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

2012 ◽  
Vol 23 (11) ◽  
pp. 2092-2108 ◽  
Author(s):  
Yuliya I. Petrova ◽  
MarthaJoy M. Spano ◽  
Barry M. Gumbiner
Keyword(s):  
Cell Surface ◽  
Conformational Changes ◽  
Binding Sites ◽  
Calcium Binding ◽  
Cell Types ◽  
P120 Catenin ◽  
Physiological Regulation ◽  
Conformational Epitopes ◽  
Calcium Binding Sites ◽  
E Cadherin

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin–catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor–induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.

scholarly journals Characteristics and Prognosis of Antibody Non-Responders with COVID-19

2021 ◽  
Author(s):  
JunYu Ding ◽  
Changxin Liu ◽  
Zhao Wang ◽  
Hua Guo ◽  
Kan Zhang ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Negative Group ◽  
Positive Group ◽  
Specific Antibodies ◽  
Reactive Protein ◽  
Virus Clearance ◽  
Cell Counts ◽  
Single Center Study ◽  
Polymerase Chain ◽  
White Blood Cell Counts

Abstract Background：The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally. The information regarding the characteristics and prognosis of antibody non-responders with COVID-19 is scarce.Method: In this retrospective, single-center study, we included all the patients with confirmed COVID-19 using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) admitted to the Fire God Mountain hospital from February 3, 2020, to April 14, 2020. A total of 1921 patients were divided into the antibody-negative group (n=94) and antibody-positive group (n=1827), and the 1:1 propensity score matching (PSM) was used to match two groups.Results: In the antibody negative group, 40 patients (42.6%) were male, 54 patients (57.4%) were female, and 49 patients (52.1%) were older than 65 years old. Cough was the most common symptoms in the antibody negative group. White blood cell counts (WBC) 6.6×109/L [5.0, 9.1], Neutrophils 4.3×109/L [3.1, 6.6], C-reactive protein 7.3 mg/L [1.3, 49.0], Procalcitonin (PCT) 0.1 ng/mL [0.0, 0.2], Interleukin-6 (IL-6) 64.2 [1.5, 28.7], Lactate dehydrogenase (LDH) 193.8 U/L [154.9,260.6], Creatine kinase 60.5 U/L [40.5, 103.7], Creatine kinase isoenzyme 10.3 ng/mL [8.2, 14.5], Urea nitrogen 5.3 mmol/L [4.0, 8.7] and Creatinine 77.7 μmol/L [60.6, 98.7] were significantly higher in antibody negative patients than in antibody positive group (P<0.005). The days of nucleic acid negative conversion in the antibody negative group was shorter than that in the antibody positive group (P < 0.001). Meanwhile, the hospitalization time of antibody negative patients was shorter than that of antibody positive patients (8.0 [6.0, 10.0] VS 13.0 [8.2, 23.0], P < 0.001).Conclusion: Some COVID-19 patients without specific antibodies had mild symptoms, but the inflammatory reaction caused by innate clinical immunity was more intense than those with antibodies, and the virus was cleared faster. The production of specific antibodies was unnecessary for SARS-CoV-2 clearance, and non-specific immune responses played an essential role in virus clearance.

Immunochemical determination of CK-MB isoenzyme in human serum. II. An enzymic approach.

1982 ◽  
Vol 28 (1) ◽  
pp. 54-58 ◽  
Author(s):  
R Wicks ◽  
M Usategui-Gomez ◽  
M Miller ◽  
M Warshaw
Keyword(s):  
Creatine Kinase ◽  
Human Serum ◽  
Adenylate Kinase ◽  
Specific Antibodies ◽  
B Subunit ◽  
Immunochemical Determination ◽  
Agarose Electrophoresis

Abstract A novel immunochemical technique for a specific enzymic determination of the myocardial isoenzyme of creatine kinase, CK-MB, involves determination of B-subunit activity of a specimen in which the M-subunit activity has been inhibited by specific antibodies to the M-subunit. Interfering activities from CK-BB isoenzyme, atypical forms of creatine kinase, and adenylate kinase are eliminated by using a blank tube in which all the M-subunit-containing isoenzymes have been removed by a specific immunoprecipitation step. The assay is convenient, linear, and reproducible, and results compare well with those by agarose electrophoresis.

INTERACTION OF PLASMIN WITH ALPHA-2 MACROGLOBULIN (α2 M): EFFECT OF ANTIFIBRINOLYTIC AGENTS

1987 ◽  
Author(s):  
J Steiner ◽  
D Strickland
Keyword(s):  
Rate Constant ◽  
Conformational Changes ◽  
Binding Sites ◽  
Second Order ◽  
Kinetic Constants ◽  
Association Rate ◽  
Antifibrinolytic Agents ◽  
Nonspecific Proteolysis ◽  
Kinetics Of ◽  
Plasmin Inhibitor

Harpel (Harpel, P.C. (1981) J. Clin. Invest 68, 46-55) reported that levels of α2M-plasmin complexes are elevated in patients receiving urokinase. He found that the distribution of plasmin between the two inhibitors, α2M and α2-plasmin inhibitor (α2PI) is dependent upon whether plasmin is added directly to plasma, or whether plasminogen in plasma is activated to plasmin by urokinase. In order to investigate possible mechanisms regulating the distribution of plasmin between these two inhibitors, a study was initiated to examine the effects of antifibrinolytic agents on the reaction of plasmin with α2M. The kinetics of the reaction were measured by monitoring conformational changes in the inhibitor resulting from exomplex formation. In order to minimize nonspecific proteolysis of the inhibitor by plasmin, the reaction was performed under conditions where the concentration of α2M was greater than that of the enzyme. The reaction between Lys77-plasmin and α2M followed second order kinetics with a rate constant of 1.8 X 105M-1 s-1. This rate was not affected 1 mM EACA or by 10 uM histidine rich glycoprotein (HRG). Further, it was found that the rate of Val442-plasmin was essentially the same as that found for Lys77-plasmin. Therefore, the binding of these ligands to the lysine binding sites of plasmin do not affect the association rate between plasmin and α2M. This is in contrast to the reaction of plasmin with α2-PI, where the binding of ligands to the lysine binding sites of plasmin reduce the rate of the reaction (Petersen & Clerrmensen (1981) Biochem. J. 199, 121-127). The kinetic constants measured predict that under conditions when the lysine binding sites of plasmin are occupied, α2M will effectively compete with α2PI in inhibiting plasmin. Further, these studies inplicate HRG as a molecule capable of regulating the distribution of plasmin between these two inhibitors.

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions

2002 ◽  
Vol 80 (4) ◽  
pp. 427-434 ◽  
Author(s):  
Wen-bin Ou ◽  
Ri-Sheng Wang ◽  
Hai-Meng Zhou
Keyword(s):  
Creatine Kinase ◽  
Dimethyl Sulfoxide ◽  
Conformational Changes ◽  
Sodium Dodecyl ◽  
Rabbit Muscle ◽  
Hydrophobic Surfaces ◽  
Muscle Creatine Kinase ◽  
Organic Mixture ◽  
Dmso Concentration ◽  
Partial Unfolding

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (<65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water–organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.Key words: creatine kinase, dimethyl sulfoxide, denaturation, activity, conformation.

Sign in / Sign up

Export Citation Format

Share Document