scholarly journals Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

1982 ◽  
Vol 203 (1) ◽  
pp. 293-298 ◽  
Author(s):  
Li-min Hsiung ◽  
A. Neil Barclay ◽  
Malcolm R. Brandon ◽  
Edith Sim ◽  
Rodney R. Porter
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Chain Structure ◽  
Affinity Column ◽  
Human Complement ◽  
Antibody Affinity ◽  
Complement Control Protein ◽  
Specific Affinity ◽  
Control Protein

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation

2008 ◽  
Vol 82 (7) ◽  
pp. 3283-3294 ◽  
Author(s):  
Viveka Nand Yadav ◽  
Kalyani Pyaram ◽  
Jayati Mullick ◽  
Arvind Sahu
Keyword(s):  
Hot Spots ◽  
Classical Pathway ◽  
Variola Virus ◽  
Amino Acid Residues ◽  
Human Complement ◽  
Binding Ability ◽  
Complement Control Protein ◽  
Functional Advantage ◽  
Complement Regulator ◽  
Control Protein

ABSTRACT Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

scholarly journals Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b

2004 ◽  
Vol 78 (17) ◽  
pp. 9446-9457 ◽  
Author(s):  
John Bernet ◽  
Jayati Mullick ◽  
Yogesh Panse ◽  
Pradeep B. Parab ◽  
Arvind Sahu
Keyword(s):  
Vaccinia Virus ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Accurate Determination ◽  
Factor H ◽  
Human Complement ◽  
Target Proteins ◽  
Complement Control Protein ◽  
Complement Proteins ◽  
Control Protein

ABSTRACT The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

scholarly journals Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Y Fujimura ◽  
Y Usami ◽  
K Titani ◽  
K Niinomi ◽  
K Nishio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Analysis ◽  
Disulfide Bonds ◽  
Platelet Glycoprotein ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

scholarly journals Characterization of primary amino acid sequence of human complement control protein factor I from an analysis of cDNA clones

1987 ◽  
Vol 242 (3) ◽  
pp. 849-856 ◽  
Author(s):  
C F Catterall ◽  
A Lyons ◽  
R B Sim ◽  
A J Day ◽  
T J R Harris
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Light Chain ◽  
Cdna Clones ◽  
Serine Proteinases ◽  
Human Complement ◽  
Factor I ◽  
Protein Factor ◽  
Control Protein

A cDNA clone of the mRNA coding for the human complement system control protein Factor I has been isolated. The predicted amino acid sequence obtained from the DNA sequence demonstrates a protein consisting of a heavy chain (Mr 35,400) linked to a light chain (Mr 27,600), both of which contain three sites for N-linked glycosylation. The light chain has clear homology with other serine proteinases, most notably in the region of the catalytically active and structurally important amino acids and shares some of the features characteristic of the plasminogen activators. The heavy chain has a clear ‘mosaic’ nature typical of the plasma serine proteinases; in particular it contains class A and class B LDL (low-density lipoprotein) receptor repeats with conserved cysteine residues similar to those found in other complement proteins.

scholarly journals SP105ATYPICAL HEMOLYTIC UREMIC SYNDROME AND C3 GLOMERULOPATHY DUE TO MONOCLONAL ANTIBODY AGAINST COMPLEMENT CONTROL PROTEIN FACTOR H

2019 ◽  
Vol 34 (Supplement_1) ◽  
Author(s):  
Paraskevi Eva Andronikidis ◽  
Papanikolaou Vasiliki ◽  
Plavoukou Styliani ◽  
Tsouka Glykeria ◽  
Delibasi Sosanna ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Hemolytic Uremic Syndrome ◽  
Factor H ◽  
C3 Glomerulopathy ◽  
Complement Control Protein ◽  
Protein Factor ◽  
Uremic Syndrome ◽  
Control Protein

scholarly journals Stability of Segments of Rabbit a-Tropomyosin

1977 ◽  
Vol 30 (6) ◽  
pp. 527 ◽  
Author(s):  
EF Woods
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Optical Rotation ◽  
Chain Structure ◽  
Cysteine Residue ◽  
Molecular Weight Determination ◽  
Cleavage Reaction ◽  
Mild Conditions ◽  
Considerable Loss

Rabbit IX-tropomyosin was cleaved into two pieces at the cysteine residue of each chain. The products were separated by chromatography and characterized by amino acid analysis, molecular weight determination in benign and denaturing solvents, optical rotation and circular dichroism. When the cleavage reaction was carried out under mild conditions which preserve the two-chain structure there was considerable loss of IX-helix in each segment.

scholarly journals 67 kDa calcimedin, a new Ca2+-binding protein

1986 ◽  
Vol 238 (1) ◽  
pp. 49-54 ◽  
Author(s):  
P B Moore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Binding Site ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Affinity Column ◽  
Binding Studies ◽  
High Affinity

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.

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