RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

SummaryThe kinetic and fibrinolytic properties of a reversibly acylated stoichiometric complex between human plasmin and recombinant staphylokinase (plasmin-STAR complex) were evaluated. The acylation rate constant of plasmin-STAR by p-amidinophenyl-p’-anisate-HCI was 52 M-1 s-1 and its deacylation rate constant 1.2 × 10-4 s-1 (t½ of 95 min) which are respectively 50-fold and around 3-fold lower than for the plasmin-streptokinase complex. The acylated complex was stable as evidenced by binding to lysine-Sepharose. However, following an initial short lag phase, the acylated plasmin-STAR complex activated plasminogen at a similar rate as the unblocked complex, whereas the acylated plasmin-streptokinase complex did not activate plasminogen. These findings indicate that STAR, unlike streptokinase, dissociates from its acylated complex with plasmin in the presence of excess plasminogen. In agreement with this hypothesis, the time course of the lysis of a 125I-fibrin labeled plasma clot submerged in citrated human plasma, is similar for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR (50% clot lysis in 2 h requires 12 nM of each agent). The plasma clearances of STAR-related antigen following bolus injection in hamsters were 1.0 to 1.5 ml/min for acylated plasmin-STAR, unblocked plasmin-STAR and free STAR, as a result of short initial half-lives of 2.0 to 2.5 min.The dissociation of the anisoylated plasmin-STAR complex and its consequent rapid clearance suggest that it has no apparent advantages as compared to free STAR for clinical thrombolysis.

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Synthesis, crystal and molecular structure, and vibrational spectra of the complex (COOH)2•2H2O•18-crown-6

Canadian Journal of Chemistry ◽

10.1139/v86-025 ◽

1986 ◽

Vol 64 (1) ◽

pp. 142-147 ◽

Cited By ~ 9

Author(s):

Suzanne Deguire ◽

François Brisse ◽

Jacques Ouellet ◽

Rodrigue Savoie

Keyword(s):

Molecular Structure ◽

Hydrogen Bonds ◽

Oxalic Acid ◽

Raman Spectra ◽

Vibrational Spectra ◽

Crystal And Molecular Structure ◽

Unit Cell ◽

Infrared And Raman Spectra ◽

O 18 ◽

Stoichiometric Complex

A stoichiometric complex of formula (COOH)2•2H2O•18-crown-6 has been obtained from oxalic acid and the macrocyclic polyether 18-crown-6. The crystals of the complex have a monoclinic unit cell and belong to the P21/c space group. The components in the adduct are linked through hydrogen bonds in a polymer-like fashion: -crown–H2O–HOOCCOOH–OH2–crown–, where the oxalic acid molecules are present in two distinct disordered orientations. The infrared and Raman spectra of the complex are also reported and interpreted.

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THE MOLECULAR WEIGHT OF RHODOPSIN AND THE NATURE OF THE RHODOPSIN-DIGITONIN COMPLEX

The Journal of General Physiology ◽

10.1085/jgp.37.3.381 ◽

1954 ◽

Vol 37 (3) ◽

pp. 381-399 ◽

Cited By ~ 143

Author(s):

Ruth Hubbard

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Extinction Coefficient ◽

Dry Weight ◽

Weight Basis ◽

Sedimentation Behavior ◽

A Value ◽

Wet Weight ◽

Single Boundary ◽

Stoichiometric Complex

The sedimentation behavior of aqueous solutions of digitonin and of cattle rhodopsin in digitonin has been examined in the ultracentrifuge. In confirmation of earlier work, digitonin was found to sediment as a micelle (D-1) with an s20 of about 6.35 Svedberg units, and containing at least 60 molecules. The rhodopsin solutions sediment as a stoichiometric complex of rhodopsin with digitonin (RD-1) with an s20 of about 9.77 Svedberg units. The s20 of the RD-1 micelle is constant between pH 6.3 and 9.6, and in the presence of excess digitonin. RD-1 travels as a single boundary also in the electrophoresis apparatus at pH 8.5, and on filter paper at pH 8.0. The molecular weight of the RD-1 micelle lies between 260,000 and 290,000. Of this, only about 40,000 gm. are due to rhodopsin; the rest is digitonin (180 to 200 moles). Comparison of the relative concentrations of RD-1 and retinene in solutions of rhodopsin-digitonin shows that RD-1 contains only one retinene equivalent. It can therefore contain only one molecule of rhodopsin with a molecular weight of about 40,000. Cattle rhodopsin therefore contains only one chromophore consisting of a single molecule of retinene. It is likely that frog rhodopsin has a similar molecular weight and also contains only one chromophore per molecule. The molar extinction coefficient of rhodopsin is therefore identical with the extinction coefficient per mole of retinene (40,600 cm.2 per mole) and the E(1 per cent, 1 cm., 500 mµ) has a value of about 10. Rhodopsin constitutes about 14 per cent of the dry weight, and 3.7 per cent of the wet weight of cattle outer limbs. This corresponds to about 4.2 x 106 molecules of rhodopsin per outer limb. The rhodopsin content of frog outer limbs is considerably higher: about 35 per cent of the dry weight, and 10 per cent of the wet weight, corresponding to about 2.1 x 109 molecules per outer limb. Thus the frog outer limb contains about five hundred times as much rhodopsin as the cattle outer limb. But the relative volumes of these structures are such that the ratio of concentrations is only about 2.5 to 1 on a weight basis. Rhodopsin accounts for at least one-fifth of the total protein of the cattle outer limb; for the frog, this value must be higher. The extinction (K500) along its axis is about 0.037 cm.2 for the cattle outer limb, and about 0.50 cm.2 for the frog outer limb.

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The mechanism of activation of NADPH oxidase in the cell-free system: the activation process is primarily catalytic and not through the formation of a stoichiometric complex

Biochemical Journal ◽

10.1042/bj3410251 ◽

1999 ◽

Vol 341 (2) ◽

pp. 251-255 ◽

Cited By ~ 26

Author(s):

Andrew R. CROSS ◽

Richard W. ERICKSON ◽

John T. CURNUTTE

Keyword(s):

Nadph Oxidase ◽

Binding Site ◽

Single Molecule ◽

Active State ◽

Stable Complex ◽

Activation Process ◽

Free System ◽

Cell Free System ◽

Flavocytochrome B ◽

Stoichiometric Complex

It is commonly assumed that activation of the superoxide-generating NADPH oxidase requires the formation of a stable complex between flavocytochrome b-245 (the gp91phox/p22phox heterodimer) and the cytosolic cofactors p47phox, p67phox and Rac2. This association is thought to convert flavocytochrome b-245, which contains the NADPH-binding site, flavin and haem centres, from an inactive into an active state. Here we provide evidence that, in the cell-free system, this activation process does not necessarily require the formation of a stable stoichiometric complex between the phox proteins. To explain this data we propose the hypothesis that p67phox (and possibly Rac2), are capable of activating flavocytochrome b-245 in a catalytic fashion, where a single molecule of p67phox (or Rac2) is capable of activating multiple flavocytochrome b-245 molecules.

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Synthesis, crystal structure, and vibrational spectra of the complex maleic acid•2H2O•18-crown-6

Canadian Journal of Chemistry ◽

10.1139/v90-334 ◽

1990 ◽

Vol 68 (12) ◽

pp. 2183-2189 ◽

Cited By ~ 8

Author(s):

Pierre Audet ◽

Rodrigue Savoie ◽

Michel Simard

Keyword(s):

Crystal Structure ◽

Water Molecule ◽

Maleic Acid ◽

Water Molecules ◽

Infrared And Raman Spectra ◽

X Ray Diffraction ◽

Monoclinic System ◽

X Ray ◽

O 18 ◽

Stoichiometric Complex

A stoichiometric complex of formula maleic acid•2H2O•18-crown-6 has been obtained from maleic acid and the macrocyclic polyether 18-crown-6. Crystals of this complex have been shown by X-ray diffraction crystallography to belong to the Cc space group of the monoclinic system. The acid molecules in the adduct are linked to each other through a water molecule, giving infinite [-acid-H2O-]n chains. They are also linked to the crown ether via water molecules. The infrared and Raman spectra of the complex are presented and compared to those of crystalline maleic acid. Keywords: maleic acid/18-crown-6, structure, X-ray, spectra.

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Coactivator functions in a stoichiometric complex with anaphase‐promoting complex/cyclosome to mediate substrate recognition

EMBO Reports ◽

10.1038/sj.embor.7400482 ◽

2005 ◽

Vol 6 (9) ◽

pp. 873-878 ◽

Cited By ~ 37

Author(s):

Lori A Passmore ◽

David Barford

Keyword(s):

Substrate Recognition ◽

Anaphase Promoting Complex ◽

Stoichiometric Complex

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Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

The Journal of Cell Biology ◽

10.1083/jcb.127.1.107 ◽

1994 ◽

Vol 127 (1) ◽

pp. 107-115 ◽

Cited By ~ 317

Author(s):

L M Machesky ◽

S J Atkinson ◽

C Ampe ◽

J Vandekerckhove ◽

T D Pollard

Keyword(s):

Gel Filtration ◽

Protein Sequences ◽

Rabbit Muscle ◽

Muscle Actin ◽

Novel Proteins ◽

Cell Extracts ◽

A Value ◽

Stokes Radius ◽

Globular Particle ◽

Stoichiometric Complex

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

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Isolation and Partial Characterization of Equine Antithrombin III.

10.1055/s-0039-1684573 ◽

1979 ◽

Cited By ~ 1

Author(s):

D.W. Estry ◽

T.G. Bell ◽

G.H. Tishkoff ◽

J.C. Mattson ◽

S.C. Estry

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Stable Complex ◽

Sds Page ◽

At Iii ◽

Human Thrombin ◽

Horse Plasma ◽

Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

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