Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.     

Fluoritisierte Radiolarien aus Kieselkalk-Bänken des Mittel-Viseums (Unterkarbon) des Rheinischen Schiefergebirges (Deutschland)

From siliceous shales (Lower Carboniferous, Rheinisches Schiefergebirge), in direct neighborhood to a bed of allodapic limestone, the following fluontized radiolarian fauna has been extracted by chemical transformation of originally calcified skeletons:Albaillella cartalla, Latentifistula turgida, Eostylodktya cf. eccentrica, Tetragregnon sycamorensis sycamorensis, Belowea variabilis, Callela? hexactinia, Entactinia tortispina and Entactinia variospina. The limestone bed has been dated by calcareous foraminifera as being mid Visean in age (V 2b-3a, Cf 5-foraminiferal zone). The diagenetic calcification took place after the selective dissolution of the skeletons and was in itself not selective.     

1H nuclear-magnetic-resonance studies of the three-dimensional structure of the cardiotoxin CTXIIb from Naja mossambica mossambica in aqueous solution and comparison with the crystal structures of homologous toxins

Using the previously reported sequence-specific 1H-NMR assignments, structural constraints for the cardiotoxin CTXIIb from Naja mossambica mossambica were collected. These include distance constraints from nuclear Overhauser enhancement measurements both in the laboratory and in the rotating frame, dihedral angle constraints derived from spin-spin coupling constants, and constraints from hydrogen bonds and disulfide bridges. Structure calculations with the distance geometry program DISMAN confirmed the presence of the previously identified antiparallel beta-sheets formed by residues 1-5 and 10-14, and by 20-27, 35-39 and 49-55, and established the nature of the connections between the individual beta-strands. These include a right-handed crossover between the two peripheral strands in the triple-stranded beta-sheet, and a type I tight turn immediately preceding the beta-strand 49-55. The spatial arrangement of the polypeptide backbone in the solution structure of CTXIIb is closely similar to that in the crystal structure of the homologous cardiotoxin VII4 from the same species. In an Appendix the origin of the large pH dependence of two amide proton chemical shifts in CTXIIb is explained.     

Molecular characterization of the hemolysin determinant of Serratia marcescens.

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.     

Enzymatic properties of proteolytic derivatives of human alpha-thrombin

The use of derivatives of alpha-thrombin obtained by limited proteolysis, that have only a single peptide bond cleaved, allowed the unequivocal correlation between the change in covalent structure and alteration of the enzymatic properties. beta T-Thrombin contains a single cleavage in the surface loop corresponding to residues 65-83 of alpha-chymotrypsin [Birktoft, J. J., & Blow, D. M. (1972) J. Mol. Biol. 68, 187-240]. Compared with alpha-thrombin, this modification had a minor effect on the following: (1) The Michaelis constant (Km) for two tripeptidyl p-nitroanilide substrates increased 2-3 fold, whereas the catalytic constant (k cat) remained unaltered. (2) A 2-3 fold increase in the binding constant (KI) of a tripeptidyl chloromethane inhibitor was observed, but the inactivation rate constant (k i) was the same, which indicated that the nucleophilicity of the active-site histidyl residue had not changed. (3) The second-order rate constant for the inhibition by antithrombin III decreased 2-fold. Heparin accelerated the inactivation, and the degree of acceleration was similar to that obtained with alpha-thrombin. Pronounced effects of the cleavage of this loop were found. (1) The cleavage of fibrinogen was approximately 80-fold slower than that with alpha-thrombin. This was mainly due to a 40-fold decrease in k cat. In contrast, only a 1.9-fold increase in the Michaelis constant was observed. (2) The affinity for thrombomodulin had decreased 39-fold compared to alpha-thrombin. epsilon-Thrombin contains a single cleaved peptide bond in the loop corresponding to residues 146-150 in alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd27+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.     

Trypsin-like activity and thromboxane release in adult respiratory distress syndrome

Plasmatic immunoreactive trypsin (IRT), thromboxane and trypsin-like enzymatic activity were measured in 117 patients at risk of developing adult respiratory distress syndrome (ARDS) (53 multiple injury, 30 abdominal surgery, 17 acute pancreatitis, 12 burnt and 5 disseminated intravascular coagulation patients). 69 of these patients developed ARDS.Immunoreactive trypsin and thromboxane were measured by radio-immuno-assay and trypsin-like enzymatic activity by spectrophotometry, using a specific chromogenic substrate.Mean IRT value was 675 ng/ml in ARDS and 265 ng/ml in non ARDS patients (p < 0.05). Mean IRT value was 685 ng/ml in sepatic and 170 ng/ml in non septic patients (p < 0.01). An abnormal trypsin-like enzymatic activity was measured in 26 ARDS patients. In 60 patients (37 ARDS and 23 non ARDS), thromboxane appeared in plasma simultaneously or about 24 hours after the beginning of IRT release. The importance of thromboxane release parallels the intensity of IRT. Originating from pancreas, trypsin can appear in plasma either by absortion from gastrointestinal tract or after pancreatic ischemia.     

Steady flow visualization in a rigid canine aortic cast

Steady flow studies were conducted in a transparent canine aortic cast. The cast segment stretched from the aortic valve to beyond the renal arteries and included all major branches. Flow was visualized by analysis of dye streaklines. Flow rates for basal and exercising cardiovascular states were simulated. The Reynolds numbers in the ascending aorta for basal and exercising conditions were 900 and 1587 respectively. Aortic core flow was laminar in basal simulations. Disturbed flow commenced in the upper descending aorta with exercising flow rates. Separation zones existed along the inner curvature of the aortic arch and the proximal walls of the brachiocephalic, left subclavian, and coeliac arteries. Such zones may exist over a portion of the cardiac cycle. If either renal artery was occluded, then a vortex formed. This vortex is associated with high shear regions which correlate well with sites where sudanophilic lesions have been reported in cholesterol-fed nephrectomized rabbits.     

Analysis of outer membrane proteins which are associated with growth of Bacteroides thetaiotaomicron on chondroitin sulfate.

By analyzing outer membrane proteins of Bacteroides thetaiotaomicron on two-dimensional polyacrylamide gels, we were able to identify 10 protein spots that were associated with growth on chondroitin sulfate but not with growth on glucuronic acid or other monosaccharides. These proteins were distinct from the outer membrane polypeptides that were associated with growth on two other negatively charged polysaccharides, polygalacturonic acid and heparin. Of the 10 protein spots that were associated with growth on chondroitin sulfate, 4 could be detected on immunoblots with antiserum that had been raised against outer membranes from bacteria grown on chondroitin sulfate and then cross-adsorbed with membranes from bacteria grown on glucose. Synthesis of these four proteins appeared to be regulated coordinately with synthesis of the two enzymes that degrade chondroitin sulfate, chondroitin lyase I and II. Although one of the four proteins (Mr 110,000) was similar in molecular weight to the chondroitin lyases, the cross-adsorbed antiserum which detected this outer membrane protein did not cross-react with either of these two enzymes.