Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour

AbstractThe far-ultraviolet circular dichroism spectra of fibrinogens phosphorylated by protein kinase C or casein kinase II indicated a conformational change corresponding to an increase in ordered secondary structure. The spectra of protein kinase A- or casein kinase I-phosphorylated fibrinogens did not differ substantially from the control. Fluorescence studies indicated changes in the tertiary structure around tryptophan residues for protein kinase A- or C-phosphorylated fibrinogens, but failed to show any such change for fibrinogen phosphorylated by either of the casein kinases. This latter result was also confirmed by circular dichroism measurements in the near-ultraviolet region. The apparent increase in ordered structure was proposed as an explanation for the slower rate of plasmin degradation seen in fibrinogens after phosphorylation by protein kinase C [6], and casein kinase II, especially as both spectral changes and plasmin degradation rate were unaffected by alkaline phosphatase

Interaction of cysteine proteinases with recombinant kininogen domain 2, expressed in Escherichia coli

AbstractThe calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli. Expression of soluble protein was optimal at 15°C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity (Ki 0.3–1 nM) for cathepsin L and papain, and a somewhat lower affinity (Ki∼ 15 nM) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated

Current and future impact of osteoarthritis on health care: a population-based study with projections to year 2032.

To estimate the current and future (to year 2032) impact of osteoarthritis (OA) health care seeking

ADAMTS13 phenotype in plasma from normal individuals and patients with thrombotic thrombocytopenic purpura

The activity of ADAMTS13, the von Willebrand factor cleaving protease, is deficient in patients with thrombotic thrombocytopenic purpura (TTP). In the present study, the phenotype of ADAMTS13 in TTP and in normal plasma was demonstrated by immunoblotting. Normal plasma (n = 20) revealed a single band at 190 kD under reducing conditions using a polyclonal antibody, and a single band at 150 kD under non-reducing conditions using a monoclonal antibody. ADAMTS13 was not detected in the plasma from patients with congenital TTP (n = 5) by either antibody, whereas patients with acquired TTP (n = 2) presented the normal phenotype. Following immunoadsorption of immunoglobulins, the ADAMTS13 band was removed from the plasma of the patients with acquired TTP, but not from that of normal individuals. This indicates that ADAMTS13 is complexed with immunoglobulin in these patients. The lack of ADAMTS13 expression in the plasma from patients with hereditary TTP may indicate defective synthesis, impaired cellular secretion, or enhanced degradation in the circulation. This study differentiated between normal and TTP plasma, as well as between congenital and acquired TTP. This method may, therefore, be used as a complement in the diagnosis of TTP

Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride Evidence for an independently unfolding structural region

AbstractFar-ultraviolet circular dichroism and tryptophan fluorescence measurements showed that the reversible unfolding of the cysteine proteinase inhibitor, chicken cystatin, by guanidinium chloride is a two-step process with transition midpoints at ≈3.4 and ≈5.4 M denaturant. The partially unfolded intermediate had both far- and near-ultraviolet circular dichroism and fluorescence emission spectra comparable to those of the native protein. The largely retained tertiary structure suggests that the intermediate represents a species in which a separate region of lower stability has been unfolded, rather than an intermediate of the ‘molten globule’ type. Such a structurally independent region is apparent in the three-dimensional structure of the inhibitor