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1.
Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions. 相似文献
2.
BS Sabna Thankappan Bency Mahendran Ramasamy Muthusamy Gayathri Femil selta Daniel Raja Angayarkanni Jayaraman 《Probiotics and antimicrobial proteins》2021,13(4):993-1004
Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of... 相似文献
3.
Anderson O. Lobo Erica F. Antunes Mariana BS Palma Cristina Pacheco‐Soares Vladimir J. Trava‐Airoldi Evaldo J. Corat 《Cell biology international》2010,34(4):393-398
Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications. 相似文献
4.
Contribution of A1 subunit residue Q316 in thrombin-activated factor VIII to A2 subunit dissociation
Blood coagulation factor VIII (fVIII) is activated by thrombin to form an A1/A2/A3-C1-C2 heterotrimer, which functions as a cofactor for factor IXa during intrinsic pathway factor X activation. Human thrombin-activated fVIII (fVIIIa) decays rapidly because of first-order dissociation of the A2 subunit, which may function to regulate the coagulation mechanism. The three fVIII A domains each consist of two cupredoxin-like subdomains. Substitution of the COOH-terminal A1 subdomain of porcine fVIIIa, which decays more slowly than human fVIIIa, reduces the dissociation rate constant for fVIIIa decay. Examination of a human fVIII A1-A2-A3 homology model [Pemberton, S., et al. (1997) Blood 89, 2413-2421) revealed a possible interaction between Q316 in the FG helix of the COOH-terminal A1 subdomain and M539 in the FG helix of the NH2-terminal A2 subdomain, which are sites where human and porcine fVIII differ. Decays of purified recombinant human and porcine fVIIIa and the human fVIIIa mutants Q316H, M539L and Q316H/M539L were compared at 23 and 37 degrees C. The decay rates of the Q316H and Q316H/M539L mutants, but not the M539L mutant, were significantly slower than human fVIIIa. These results indicate that the FG helix of the COOH-terminal A1 cupredoxin-like subdomain of fVIII may be under selective pressure by the requirements of hemostatic balance. 相似文献
5.
Jon Telling Ken Voglesonger Chelsea N. Sutcliffe Georges Lacrampe-Couloume Elizabeth Edwards Barbara Sherwood Lollar 《Geomicrobiology journal》2018,35(2):108-119
Precambrian Shield rocks host the oldest fracture fluids on Earth, with residence times up to a billion years or more. Water–rock reactions in these fracture systems over geological time have produced highly saline fluids, which can contain millimolar concentrations of H2. Mixing of these ancient Precambrian fluids with meteoric or palaeo-meteoric water can occur through tectonic fracturing, providing microbial inocula and redox couples to fuel blooms of subsurface growth. Here, we present geochemical and microbiological data from a series of borehole fluids of varying ionic strength (0.6–6.4 M) from the Thompson Mine (Manitoba) within the Canadian Precambrian Shield. Thermodynamic calculations demonstrate sufficient energy for H2-based catabolic reactions across the entire range of ionic strengths during mixing of high ionic strength fracture fluids with meteoric water, although microbial H2 consumption and cultivable H2-utilizing microbes were only detected in fluids of ≤1.9 M ionic strength. This pattern of microbial H2 utilization can be explained by the higher potential bioenergetic cost of organic osmolyte synthesis at increasing ionic strengths. We propose that further research into the bioenergetics of osmolyte regulation in halophiles is warranted to better constrain the habitability zones of hydrogenotrophic ecosystems in both terrestrial subsurface, including potential future radioactive waste disposal sites, and other planetary body crustal environments, including Mars. 相似文献
6.
The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary
tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically
removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in
both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas
the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and
CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage
of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no
significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave
the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical
markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on
the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment
of metastases.
This revised version was published online in November 2006 with corrections to the Cover Date. 相似文献
7.
8.
Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant. 相似文献
9.
V P Mutucumarana E J Duffy P Lollar A E Johnson 《The Journal of biological chemistry》1992,267(24):17012-17021
The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface. 相似文献
10.
Comparisons of the molecular evolutionary process at rbcL and ndhF in the grass family (Poaceae) 总被引:2,自引:1,他引:1
We examine rate heterogeneity among evolutionary lineages of the grass
family at two plasmid loci, ndhF and rbcL, and we introduce a method to
determine whether patterns of rate heterogeneity are correlated between
loci. We show both that rates of synonymous evolution are heterogeneous
among grass lineages and that are heterogeneity is correlated between loci
at synonymous sites. At nonsynonymous sites, the pattern of rate
heterogeneity is not correlated between loci, primarily due to an aberrant
pattern of rate heterogeneity at nonsynonymous sites of rbcL. We compare
patterns of synonymous rate heterogeneity to predictors based on the
generation time effect and the speciation rate hypotheses. Although there
is some evidence for generation time effects, neither generation time
effects nor speciation rates appear to be sufficient to explain patterns of
rate heterogeneity in the grass plastid sequences.
相似文献