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A Betz

Publications and source records attributed to A Betz.

At least 55 records · Page 3Linked to original sources
PubMed1994

Transient hydrogen bonds identified on the surface of the NMR solution structure of Hirudin.

Recombinant desulfatohirudin retains largely the thrombin-inhibitory activity of natural hirudin from Hirudo medicinalis and causes at most minimal immune response in humans. With regard to potential pharmaceutical applications it is of interest to further investigate the structural basis of hirudin functions. In this paper transient hydrogen bonds between backbone amide protons and side-chain carboxylates on the protein surface of desulfatohirudin (variant 1) have been identified using two-dimensional 1H NMR experiments and site-directed mutagenesis. The analysis of pH titration curves measured with NMR enabled the determination of the pK values of all 13 carboxylates, and downfield shifts larger than 0.2 ppm arising from weak bonding interactions with carboxylates were observed for the amide protons of Gly 25, Ser 32, Glu 35, and Cys 39. For these backbone amide protons virtually identical titration parameters were observed in intact desulfatohirudin and the mutant, truncated hirudin(1-51), demonstrating that the hydrogen bond acceptors are located in the N-terminal polypeptide segment 1-51. The hydrogen bonds Gly 25 NH-Glu 43 delta COO-, Ser 32 NH-Glu 35 delta COO-, Glu 35 NH-Asp 33 gamma COO-, Glu 35 NH-Glu 35 delta COO-, and Cys 39 NH-Glu 17 delta COO- were identified by considering spatial proximity in the NMR solution structure of hirudin(1-51), and comparing the pK values for the amide protons and the carboxylates in desulfatohirudin and the mutants hirudin(E43Q), hirudin(E35Q), hirudin(D33N) and hirudin(E17A).(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1994

Mapping of the thrombin des-ETW conformation by using site-directed mutants of hirudin. Evidence for the induction of nonlocal modifications by mutagenesis.

Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alters its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's structure [Le Bonniec, B. F., Guinto, E. R., & Esmon, C. T. (1992) J. Biol. Chem. 267, 19341-19348]. In an attempt to localize conformational change in this thrombin mutant (des-ETW), its affinity for 43 site-directed mutants of hirudin have been compared with that of native thrombin. The inhibition constants (KI) of recombinant and natural hirudin with des-ETW were found to be approximately 2300-fold higher than those with thrombin. The reduced affinity was predominantly the result of an increase in the dissociation rate constant rather than a decrease in the association rate constant. For most of the hirudin mutants tested, the difference in binding energy between thrombin and des-ETW [delta delta Gbo(IIa-ETW)] was about 19.9 kJ mol-1 and comparable to that of hirudin; in particular, all mutations in the C-terminal region of the inhibitor did not affect delta delta Gbo(IIa-ETW). Thus, the conformation of thrombin's anion-binding exosite seems unaltered by the des-ETW mutation. In contrast, hirudin substitutions involving Val1', Val2', Thr4', Asp5', and Ser19', as well as the addition of an N-terminal glycine, resulted in values of delta delta Gbo(IIa-ETW) which were 2 to 10 kJ mol-1 lower than that for hirudin. Furthermore, a Leu15'-->Ala hirudin mutant behaved with des-ETW, as a partial competitive inhibitor rather than a tight-binding inhibitor, in contrast to all other hirudin variants tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin.

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Amino Acid Sequence↗
PubMed1994

[Possibilities in the reconstruction of bone defects].

The importance of the functional unit "bone-soft-tissue" for the treatment of bone defects is discussed. A division is made between non-vascular and vascular bone transport. The individual methods are explained and their clinical significance is illustrated partly using case material. In general, the best way to fill small defects in vital soft tissue is to use cancellous autografts. For bridging longer bone defects callus distraction is the method of first choice, but in individual cases vascularized bone transfer can be taken from the iliac crest or the fibula. Homo- or heterografts should only be used in cases where no autologic bone material is available or when there is not enough.

Arm Injuries↗
PubMed1994

[Stabilizing procedures in tumor-induced bone defects of the pelvis and extremities].

With malignant tumors of the skeleton the same therapeutic limb-saving guidelines must apply for the proximal sections of the locomotor system--the shoulder and pelvic girdles--as for the limbs themselves, especially since amputation here can hardly be surpassed by other resectional interventions with respect to operative risk, disfigurement and functional loss. The possibilities of surgical therapy for tumorous invasion of the pelvic girdle and the extremities are considered in some detail.

Adolescent↗
PubMed1993

[Minimal osteosynthesis of tibial pilon fracture].

Fifty-eight patients with a total of 60 pillion fractures of the tibia were treated, mainly operatively, in the Department of Surgery (Klinikum Innenstadt, University of Munich) from 1984 to 1989. After 1-4 years, it was possible to question 90% of the patients, and 75% were also examined clinically and radiologically. The most frequent cause of fracture was a heavy impact, whereas sporting injuries accounted for only 13%. Thus, there was a high incidence (39%) of severe fractures (type C, AO classification) with tissue damage. The initial operative procedure was minimal osteosynthesis in 77% of the patients and tibial plate fixation in 20%. Delayed wound healing was observed in 17% with a rate of osteitis of 5%. The follow-up examination showed good to excellent results in 74%, and in two-thirds of the patients only minor arthrotic changes or none of all were found. Despite less severe fracture types in the group with tibial plate stabilization they more frequently had impaired wound healing (27% vs 14%) and severe posttraumatic arthrosis (43% vs 18%) than patients with minimal osteosynthesis. The results are discussed.

Adult↗
PubMed1992

Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin.

Site-specific substitutions of the first five amino acids of the thrombin inhibitor hirudin have been made and the effects of these substitutions on the kinetics of formation of the thrombin-hirudin complex evaluated. The effects of different substitutions of Val1 indicate that nonpolar interactions play a major role in the binding of this residue. In the second position (Val2), polar amino acids were better accommodated than in the first. The mutant with arginine in the second position bound particularly well to thrombin; its dissociation constant was 9-fold lower than that of wild-type recombinant hirudin. Comparison of the effects of single and double mutations involving Val1 and Val2 indicates that there was no cooperativity in the binding of these two residues. Elimination of the hydrophobic interactions made by the aromatic ring of Tyr3 of hirudin resulted in a large loss of binding energy (12.7 kJ mol-1). Replacement of Thr4 of hirudin by serine and alanine suggested that both the gamma-methyl and the hydroxyl group of the threonine were important in the stabilization of the thrombin-hirudin complex. Replacement of Asp5 of hirudin by alanine and glutamate caused about the same loss in binding energy (5 kJ mol-1). The effects of site-specific substitutions are discussed in terms of the crystal structure of the thrombin-hirudin complex. Molecular modeling provided plausible explanations for many of the observed effects. For instance, such studies suggested that the improved binding of the mutant with arginine in the second position could be due to an interaction of the arginine with the primary specificity pocket.

Amino Acid Sequence↗
PubMed1992

pH dependence of the interaction of hirudin with thrombin.

The kinetics of the inhibition of human alpha-thrombin by recombinant hirudin have been studied over the pH range from 6 to 10. The association rate constant for hirudin did not vary significantly over this pH range. The dissociation constant of hirudin depended on the ionization state of groups with pKa values of about 7.1, 8.4, and 9.2. Optimal binding of hirudin to thrombin occurred when the groups with pKa values of 8.4 and 9.0 were protonated and the other group with a pKa of 7.1 was deprotonated. The pH kinetics of genetically engineered forms of hirudin were examined in an attempt to assign these pKa values to particular groups. By using this approach, it was possible to show that protonation His51 and ionization of acidic residues in the C-terminal region of hirudin were not responsible for the observed pKa values. In contrast, the pKa value of 8.4 was not observed when a form of hirudin with an acetylated alpha-amino group was examined, and, thus, this pKa value was assigned to the alpha-amino group of hirudin. The requirement for this group to be protonated for optimal binding to thrombin is discussed in terms of the crystal structure of the thrombin-hirudin complex. Examination of this structure allowed the other pKa values of 7.1 and 9.2 to be tentatively attributed to His57 and the alpha-amino group of Ile16 of thrombin.

Hirudins↗
PubMed1992

[Diagnosis, classification and indications for surgical treatment of pelvic ring fractures].

In cases of high-energy trauma, it is well known that there is a high incidence of pelvic fractures. The mechanism of injury, inspection and physical examination of the victim at the accident site direct attention to a pelvic fracture. In most cases, the first radiological examination (A.P. X-ray of the pelvis, oblique view of the obturator and oblique view of the ilium) shows the extent of the bony lesion. The diagnosis and therapy of lesions of the urinary tract, of intra-abdominal organs and blood vessels are vitally important. For the definitive operation of unstable pelvic ring fractures, additional diagnostic means, i.e., CT scans to distinguish posterior instability, can be necessary. Osteosynthesis can only be successful in the pelvis if one has a biochmechanical understanding of the physiological flux of force from the neck of the femur via the acetabular fossa to the sacroiliac joint. The sacroiliac ligaments have a particularly important support function. For assessing stability and classifying the traumatic patterns, it is helpful to use Pennal's classification, which takes the direction of the action of force into account. Three basic forms can be distinguished: anteroposterior compression, lateral compression and vertical avulsion. Depending on the extent of the traumatic pattern, one can distinguish three subtypes: type 1 is treated conservatively while types 2 and 3 require surgical treatment. The biochmechanics, traumatic patterns, diagnostics and treatment techniques applied are described clearly and with good illustrations.

Biomechanical Phenomena↗
PubMed1992

[Pelvic girdle fractures--must they be stabilized?].

Osteosynthesis can only be successful in the pelvis if one has a biomechanical understanding of the physiological flux of force from the neck of the femur via the acetabular fossa to the sacroiliac joint. The sacroiliac ligaments have a particularly important support function here. For assessing stability and classifying the traumatic patterns it is helpful to use Pennal's classification, which takes the direction of the action of force into account. Three basic forms can be distinguished: anteroposterior compression, lateral compression and vertical avulsion. Depending on the extent of the traumatic pattern one can distinguish three subtypes. Type 1 is treated conservatively while types 2 and 3 require surgical treatment. The traumatic patterns and treatment techniques applied are described clearly and with good illustrations.

Biomechanical Phenomena↗
PubMed1991

Mechanistic studies on thrombin catalysis.

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex.

The role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex has been investigated by site-directed mutagenesis. The residues Phe56, Pro60, and Tyr63 of hirudin were replaced by a number of different amino acids, and the kinetics of the inhibition of thrombin by the mutant proteins were determined. Phe56 could be replaced by aromatic amino acids without significant loss in binding energy. While substitution of Phe56 by alanine decreased the binding energy (delta G degrees b by only 1.9 kJ mol-1, replacement of this residue by amino acids with branched side chains caused larger decreases in delta G degrees b. For example, the mutant Phe56----Val displayed a decrease in delta G degrees b of 10.5 kJ mol-1. Substitution of Pro60 by alanine or glycine resulted in a decrease in delta G degrees b of about 6 kJ mol-1. Tyr63 could be replaced by phenylalanine without any loss in binding energy, and replacement of this residue by alanine caused a decrease of 2.2 kJ mol-1 in delta G degrees b. Substitution of Tyr63 by residues with branched side chains resulted in smaller decreases in delta G degrees b than those seen with the corresponding substitutions of Phe56; for example, the mutant Tyr63----Val showed a decrease in binding energy of 5.1 kJ mol-1. The effects of the mutations are discussed in terms of the crystal structure of the thrombin-hirudin complex.

Amino Acid Sequence↗
PubMed1991

Ionic interactions in the formation of the thrombin-hirudin complex.

Site-directed mutagenesis has been used to examine the importance of each of the acidic C-terminal residues of hirudin in the formation of its complex with alpha-thrombin. The contribution to binding energy of acidic residues in the 11 C-terminal amino acids varied from 2.3 to 5.9 kJ.mol-1. The differences between the contributions of individual residues were smaller than would be expected from the crystal structures of the thrombin-hirudin complex. In particular, the small effect (2.4 kJ.mol-1) for the replacement of Asp-55 was surprising in view of the two salt bridges made by this residue. The results of studies involving multiple mutations indicated that the additivity of the effects varied with the position of the mutation. Whereas the effect of mutations involving the glutamic acid residues at positions 61 and 62 were additive, non-additivity was observed with the glutamic acid residues at positions 57 and 58.

Amino Acid Sequence↗
PubMed1991

[Traumatology in the elderly. Problems exemplified by femoral neck fractures].

The most frequent complications in response to a fracture of the femoral neck, a typical injury of the elderly, are necrosis of the head of femur and pseudoarthrosis of the femoral neck. Only the around dressed down fracture can be conveyed to conservative treatment. The classification of AO takes these problems into consideration. One possibility to preserve the femur head is by operative insertion of a dynamic hip screw (DHS). Specific problems of the diverse types of fracture and their surgical details are taken into special account.

Aged↗
PubMed1991

[Knee joint hemarthrosis. An absolute indication for operation?].

The degree of severity of an injury of the knee joint is estimated mainly on the basis of the history given by the patient, stability testing and hemarthrosis. Once these are known the surgeon usually decides whether an arthroscopy should be done or not. In a group of 365 patients who had undergone arthroscopy after acute knee injury, we made a retrospective check of the indications. The purpose of the study was to evaluate the sensitivity of hemarthrosis as an indicator of severe injury of the knee joint necessitating operative treatment. For stability testing we used the varus and valgus stress test, the Lachman test, the Anterior drawer test, the pivot shift test and the anterior drawer test with medial and lateral rotation. The Lachman test was repeated under general anesthesia in the majority of patients just before the arthroscopy. The results were evaluated retrospectively with the aid of electronic data processing. In nearly 70% of cases the arthroscopy was indicated because of positive signs of instability together with a typical history provided by the patient or hemarthrosis. In another 27%, the hemarthrosis was the sole reason for the surgeon's decision to perform an arthroscopic investigation of the injured knee joint. In 80.5% of all cases the clinical diagnosis of lesion of the anterior cruciate ligament (ACL) or combined injury to ligaments and menisci was confirmed by the arthroscopy. In addition to this group, in another 10.8% we found other severe lesions necessitating by operation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗