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R Berendes

Publications and source records attributed to R Berendes.

13 recordsLinked to original sources
PubMed1994

Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study.

We have used electron microscopy to analyse the structure of wild-type human annexin V (recombinant and placental) and of several mutants (single and double point mutants) bound to monolayers composed of DOPS, DOPE, or brain extract (Folch fraction III). On these phospholipids and on DOPS/DOPC (3:1, w/w) protein trimers, as also found in 3-D crystals, assemble to form a hexagonal lattice with a unit vector length of about 18 nm. The resolution obtained in projection is 1.7 to 2.2 nm for wild-type and mutants. There are no significant differences between the annexin V mutants and the wild-type protein at this resolution. All proteins bind as trimers with their convex side harbouring the Ca(2+)-binding sites facing the membrane. A comparison of the 3-D reconstruction of annexin V wild-type with the high resolution crystal structure shows that the domain structure is preserved but the relative orientation of the modules (II/III) and (I/IV) is slightly changed so that the Ca(2+)-binding sites in all four domains (including the recently observed binding site in domain III) become coplanar to the membrane. The thickness of the molecule obtained in the 3-D reconstruction corresponds well with the thickness of the high resolution crystal structure indicative of peripheral binding of annexin V without substantial penetration of the membrane.

Annexin A5↗
PubMed1994

Structural and electrophysiological analysis of annexin V mutants. Mutagenesis of human annexin V, an in vitro voltage-gated calcium channel, provides information about the structural features of the ion pathway, the voltage sensor and the ion selectivity filter.

Annexin V binds to phospholipids in a calcium-dependent manner and exhibits calcium channel activity in vitro. We prepared a variety of mutants yielding information about the structure-function relationship of the ion channel activity. All mutants were characterized by X-ray crystallography, electron microscopy and electrophysiological measurements. Their structures are insignificantly changed whereas their electrophysiological properties are drastically different. Glu95, located in the central hydrophilic pore of the molecule, is crucial for the ion selectivity filter as its exchange leads to reduced calcium and increased sodium conductance. The removal of Glu17, located on the protein surface and far from the ion conduction pathway, leads to the appearance of a second conductance level of 9 pS in addition to the conductance level of about 30 pS in the wild-type molecule. This was also the case for Glu78, which is part of a weak calcium binding site. The exchange of Glu17 and Glu78 produced a mutant retaining only the smaller conductance level. We conclude that these two residues influence the angle between the two halves of the molecule, which determines the diameter of the ion conduction pathway, thereby leading to the occurrence of a second conductance level.

Amino Acid Sequence↗
PubMed1994

Annexin V: the key to understanding ion selectivity and voltage regulation?

Annexin V is a Ca(2+)-dependent membrane-binding protein that forms voltage-dependent Ca2+ channels in phospholipid bilayers and is the first ion channel to be structurally and functionally characterized. Data outlined here indicate that key amino acid residues act as selectivity filters and voltage sensors, thereby regulating the permeability of the channel pore to ions.

Annexin A5↗
PubMed1993

Structure-function analysis of the ion channel selectivity filter in human annexin V.

Electrophysiology and structural studies were performed on an annexin V variant containing a mutation of glutamic acid-95 to serine in the center of the pore region. The mutation resulted in a lower single channel conductance for calcium and a strongly increased conductance for sodium and potassium, indicating that glutamic acid-95 is a crucial constituent of the ion selectivity filter. There were only minor differences in the crystal structures of mutant and wild-type annexin V around the mutation site; however, the mutant showed structural differences elsewhere, including the presence of a calcium binding site in domain III unrelated to the mutation. Analysis of the membrane-bound form of annexin V by electron microscopy revealed no differences between the wild type and mutant.

Annexin A5↗
PubMed1993

A rapid and efficient purification method for recombinant annexin V for biophysical studies.

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.

Animals↗
PubMed1993

Calcium influx through annexin V ion channels into large unilamellar vesicles measured with fura-2.

A new assay for the calcium channel activity of annexin V was developed. The calcium-sensitive fluorescence indicator, fura-2, was incorporated into large unilamellar vesicles (LUV). After establishing a calcium gradient across the liposomal membranes, native or mutated annexin V was added. The resulting calcium influx into the LUV detected through the fluorescence changes of fura-2 was used as a qualitative test for the electrophysiological properties of annexin V.

Annexin A5↗
PubMed1992

Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.

Amino Acid Sequence↗
PubMed1992

Short-chain dolichols of defined chain length as cofactors in reactions of the microsomal dolichyl-phosphate cycle and transglycosylations.

1) The biological cofactor and carrier activities of dolichyl phosphates of low isoprene multiplicity (n) and defined geometry, (synthesized according to L. Jaenicke and H.-U. Siegmund, Chem. Phys. Lipids 51 (1989) 159-170), were assayed in different transfer reactions of the microsomal dolichyl-phosphate cycle against natural pig liver dolichyl phosphate (n = 18 to 20). 2) The apparent Michaelis constants and maximal velocities were determined from initial reaction rates for the transfer from GDP-mannose, UDP-N-acetylglucosamine, and UDP-glucose to the synthetic truncated dolichyl phosphates. They afford quantitative comparison and show increasing biological activities from dolichyl-6 phosphate to dolichyl-11 phosphate, which is about as active as the natural mixture. This is in accord with previous findings on the starting reactions of the cycle. 3) Truncated dolichyl diphosphochitobioses, biosynthesized in vitro from synthetic dolichyl phosphates, were used as acceptors for nucleoside diphosphohexoses in solubilized membranes. All of them show about the same activity. The kinetics and yield were determined for each of the transfers. Activity is increased by adding UDP-glucose. The inactive very short-chain dolichol compounds do not interfere with the transfer to active longer chain dolichols. 4) The oligosaccharides produced by transfer of mannose and glucose to truncated dolichyl diphosphate-bound chitobiose were isolated and analysed for sugar multiplicity. The heptasaccharide and the un-decasaccharide are accumulated most, pointing to the transport across the endoplasmic membranes (ER) as the rate limiting reaction. 5) The truncated dolichyl-diphosphate-bound oligosaccharides are transferred to protein(s) by the crude, solubilized microsomal preparation independent of chain length of the cofactor/carrier, yet with increasing yield as shown by enzyme immunoblot analysis.

Animals↗
PubMed1992

Annexin V-crystal structure and its implications on function.

Annexins constitute a family of cytosolic, water soluble proteins, which bind to negatively charged phospholipids in a calcium-dependent manner. They display structural and functional features of both soluble and integral membrane proteins. The annexins face the hydrophilic as well as the hydrophobic phase (Janus-faced proteins) and mediate ion transport in vitro. We present the refined structure and molecular model of annexin V at 2.0 A resolution. The molecule is almost entirely alpha-helical, and each of the four repeats of annexin V is folded into a compact domain of similar structure. The four domains are arranged in an almost planar, cyclic array. In the center of the molecule, one can find a prominent hydrophilic pore, which we associate with the calcium-selective channel found in annexin V. Annexin V has an overall flat, slightly curved shape with two faces, one convex and one concave. The three calcium binding sites Ca1 to Ca3, all located at the convex face of the molecule, are assumed to be phospholipid binding sites, as suggested by their structural similarity to the calcium site of phospholipase A2. Soluble and membrane-bound annexin have closely similar structures, as shown by electron microscopic analysis. Several other observations provide evidence that the membrane-anchoring region of the annexin V molecule is located on the convex face. In the last part of this article, the electrophysiology of the annexins is described. Ion permeation occurs in discrete conductance states and is regulated by voltage across the membrane. A model for the annexin V-membrane interaction, the ion channel formation, and the ion conduction pathway is proposed.

Amino Acid Sequence↗
PubMed1978

[The value of the postcoital test (Sims-Huhner test) in the treatment of sterility].

The analysis of 751 postcoital tests performed in patients consulting the infertility clinic of the Department of Obstetrics and Gynecology, University of Cologne, allows the following diagnostic and prognostic conclusions: 1. In case motile spermatozoa are present in the cervical mucus, the assumption can be made that adaequate insemination takes place. 2. The postcoital test and the simultaneous examination of the cervix factors offer themselves for the assessment of the estrogenic status during the normal cycle or after stimulation with clomiphene, HMG-HCG or estrogen. 3. The timing of the postcoital test itself results in a remarkable increase in pregnancy rates. 4. The pregnancy rates are higher when higher numbers of motile sperms are found in the cervical mucus. 5. Negative or doubtful postcoital tests do not rule out the possibility of pregnancy. 6. The postcoital test provides some information about the number of spermatozoa, their progressive motility, their morphology and about the interaction of spermatozoa with the cervical mucus. However, the postcoital test cannot replace a complete semen analysis.

Cervix Mucus↗
PubMed1978

[The significance of prenatal diagnosis of twins (author's transl)].

By means of ultrasound and fetal electrocardiography the diagnosis of twin pregnancies can be made prenatally without any reservation; the diagnosis is even possible in the first half of pregnancy. Comparative analysis of two approximately equal collectives of in all 602 twin pregnancies--1,45% of all births in the years 1955-1975 at the University Clinic for Gynecology and Obstetrics of the University of Cologne--revealed a better prognosis concerning the course of pregnancy and the new-born by prenatal diagnosis. The earliest possible diagnosis of twins, resulting in more intense clinical observation--and occasional clinical treatment--facilitated a significantly longer duration of pregnancy, leading to a higher percentage of mature twins. Whereas the total number of obstetric operations was reduced because of prenatal diagnosis, selection of highrisk cases led to a distinct increase in Caesarian sections. The prolongation of pregnancy, the higher birth weights and better maturity attained, were accompanied by decrease in perinatal mortality and in improvement in the Apgar-index of the newborn in that collective, in which prenatal diagnosis was achieved.

Apgar Score↗