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Biomedical subjects

B Steipe

Publications and source records attributed to B Steipe.

24 records · Page 2Linked to original sources
PubMed1992

Refined crystal structure of a recombinant immunoglobulin domain and a complementarity-determining region 1-grafted mutant.

We report the solution of the crystal structure of a mutant of the immunoglobulin VL domain of the antibody McPC603, in which the complementarity-determining region 1 segment is replaced with that of a different antibody. The wild-type and mutant crystal structures have been refined to a crystallographic R-factor of 14.9% at a nominal resolution of 1.97 A. A detailed description of the structures is given. Crystal packing results in a dimeric association of domains, in a fashion closely resembling that of an Fv fragment. The comparison of this VL domain with the same domain in the Fab fragment of McPC603 shows that the structure of an immunoglobulin VL domain is largely independent of its mode of association, even in places where the inter-subunit contacts are not conserved between VL and VH. In all three complementarity-determining regions we observe conformations that would not have been predicted by the canonical structure hypothesis. Significant differences between the VL domain dimer and the Fab fragment in the third complementarity-determining region show that knowledge of the structure of the dimerization partner and its exact mode of association may be needed to predict the precise conformation of antigen-binding loops.

Amino Acid Sequence↗
PubMed1990

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli.

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.

Chromatography, Affinity↗
PubMed1987

In situ studies of distal convoluted tubule in rat. II. K secretion.

Microperfusion and free-flow micropuncture studies were performed in anesthetized rats to compare the rates of K flux in early and late segments of the distal tubule. Early distal segments were located within the initial 40% of distal tubule length, and late distal segments were located within the terminal 35% of the distal tubule. In early distal segments of control rats, significant K secretion was observed at perfusion rates of 6 and 14 nl/min. In high-K rats significant K secretion was only observed at the lower perfusion rate. Late segments of control rats secreted 70.5 +/- 8.2 at the low flow rate and 139 +/- 18.9 pmol X min-1 X mm-1 at the high flow rate. In K-adapted rats, values were 134.4 +/- 30.2 and 178 +/- 29.3 pmol X min-1 X mm-1. During free flow, we observed a K flux in control rats of 11.0 +/- 9.4 pmol X min-1 X mm-1 (NS) in early segments and a K flux of 55 +/- 6.5 pmol X min-1 X mm-1 in late segments. In K-adapted rats, values were 18.3 +/- 6.7 (P less than 0.05) in early and 123 +/- 17.2 pmol X min-1 X mm-1 in late segments. The majority of our data suggests that the distal convoluted tubule secretes K at a low rate that is not influenced by flow rate or a high-K diet.

Animals↗
PubMed1984

Inactivation of atrial natriuretic substance by kallikrein.

To further characterize the properties of the potent natriuretic and diuretic substance that can be extracted from atrial tissue, we investigated its susceptibility to inactivation by kallikrein and other proteolytic enzymes. Extracts of rat atrial tissue (tissue wet wt 100 mg/ml) were incubated with enzymes under standard conditions and tested by injection into nondiuretic anesthetized rats. One hour of incubation at 37 degrees C with pure porcine pancreatic kallikrein at concentrations of 250 micrograms/ml or greater significantly reduced the activity of atrial natriuretic substance. The reduction in activity was dependent on both enzyme concentration and time of incubation. The kallikrein-catalyzed degradation was completely blocked by aprotinin but was only partially retarded by soybean trypsin inhibitor. Trypsin reduced natriuretic and diuretic activity of extracts at concentrations of 400 micrograms/ml or greater, with nearly complete inactivation at a concentration of 1,000 micrograms/ml. Carboxypeptidase B also caused a concentration-dependent inactivation of the natriuretic material. Last, alpha-chymotrypsin (1,000 micrograms/ml) and elastase (1,000 micrograms/ml) were found to destroy the natriuretic activity. In a separate set of experiments natriuretic activity was observed to be retained by a 1,000 mol wt cutoff membrane. Inactivation of the natriuretic peptide by renal kallikrein is a possible mechanism for in vivo regulation of natriuretic activity.

Animals↗
PubMed1982

Micropuncture studies of the renal effects of atrial natriuretic substance.

Micropuncture studies of the renal effects of atrial natriuretic substance. Injection of atrial extract produced by homogenization, boiling and centrifugation of atrial tissue from one heart caused a 10fold increase in urine flow rate and a 30-fold increase in Na excretion. Similarly prepared extracts of ventricle were without effect. To identify the site of action of atrial natriuretic substance, extract was infused intravenously at rates corresponding to 3 or 6 atria per hour. During infusion at a rate of 3 atria per hour mean urine flow increased from 9.5 +/- 2.8 to 17.2 +/- 1.2 microliter/min and Na excretion from 0.14 +/- 0.06 to 1.78 +/- 0.14 mumol/min. Glomerular filtration rate (GFR), single nephron filtration rate (SNGFR) and proximal and loop of Henle fluid absorption did not change significantly. During infusion of 6 atria per hour, paralleling a greater rise in urine flow rate (from 6.4 +/- 2.09 to 40.3 +/- 7.5 microliter/min) and in sodium excretion (from 0.18 +/- 0.0008 to 5.97 +/- 0.93 mumol/min), filtration rate, measured for either the single nephron or the whole kidney, rose. As a consequence of the rise in GFR, delivery of fluid and chloride into the distal tubule increased significantly. These data suggest that to a major extent the natriuresis is caused by transport inhibition along collecting tubules and collecting ducts. In addition, at high doses a rise in filtration rate contributes to the natriuretic effect of atrial extracts.

Animals↗