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Biomedical subjects

W Stöcker

Publications and source records attributed to W Stöcker.

At least 37 records · Page 2Linked to original sources
PubMed1994

[Stiff-man syndrome. Report of 4 cases].

Four patients are presented who exhibited progressive muscular rigidity in both legs, the thoracolumbar, paraspinal and the abdominal muscles. In only one patient, there was an initial involvement of the shoulder girdle muscles. Electromyography in all four patients at rest recorded continuous electric activity resembling an interference pattern. Following sudden exteroceptive stimuli the activity increased in amplitude and density this corresponded clinically to painful spasms. All patients had oligoclonal banding in CSF. In the serum and CSF of three patients IgG subtype autoantibodies were detected. These have been found to be directed against GABAergic nerve terminals in the rat and human cerebellum and, more specifically, to glutamic acid decarboxylase. All patients improved on clonazepam. Trials of intermittent high-dose methylprednisolone administration gave relief from rigidity in one patient and permitted reduction of clonazepam in another. Intravenous immunoglobulins however had no effect in one patient.

Adult↗
PubMed1993

Implications of the three-dimensional structure of astacin for the structure and function of the astacin family of zinc-endopeptidases.

Astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358, 164-167]. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family. Computer-assisted modelling of the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered. The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins. Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.

Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1.8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0.158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure, the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long alpha-helices, one centrally located and one more peripheral, and of a five-stranded pleated beta-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-site distinguish their structures from astacin. The five-stranded beta-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central alpha-helix and the zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and His142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases.

Astacin, a digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype for the 'astacin family', which includes mammalian metallo-endopeptidases and developmentally regulated proteins of man, fruitfly, frog and sea urchin. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.

Amino Acid Sequence↗
PubMed1992

The 70 kD heat shock protein (hsp 70) in soil invertebrates: a possible tool for monitoring environmental toxicants.

The expression of hsp 70 after heat shock or exposure to heavy metals/molluscicides was investigated by fluorography or immunoblot in three diplopods (Glomeris marginata, Cylindroiulus punctatus, Tachypodoiulus niger), two slugs (Deroceras reticulatum, Arion ater), and one isopod (Oniscus asellus). In O. asellus, hsp 70 expression occurred after heat shock and also after lead treatment, whereby a solution of 100 mg/kg Pb2+ was sufficient. Animals of the same species taken from a heavy metal polluted site in the vicinity of a lead/zinc smelter also showed the presence of hsp 70. The comparison of laboratory and field experiments demonstrated the suitability of O. asellus for monitoring tests. In contrast, the blot pattern after contamination with 1,000 mg/kg Pb2+ (in the mentioned diplopods) or different concentrations of the molluscicide Cloethocarb (BASF, FRG) (slugs) showed no differences compared to the respective control group.

Animals↗
PubMed1991

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.

Amino Acid Sequence↗
PubMed1991

Kinetics of nitroanilide cleavage by astacin.

The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Fluorescent oligopeptide substrates for kinetic characterization of the specificity of Astacus protease.

The design of fluorescent N-dansylated oligopeptides based on the tubulin cleavage pattern by Astacus protease yields substrates that are turned over up to 10(5) times faster than those presently available. On the basis of this study, an optimal substrate for Astacus protease contains seven or more amino acids and minimally requires at least five amino acids. Direct examination of the formation and breakdown of the ES complex shows its formation occurs within milliseconds at 25 degrees C. The best heptapeptide substrate, Dns-Pro-Lys-Arg-Ala-Pro-Trp-Val, is cleaved only between the Arg-Ala (P1-P1') bond with kinetic parameters kcat = 380 s-1 and Km = 3.7 x 10(-4) M. The presence of Lys or Arg in the P1 and P2 positions yields high-turnover substrates. In the P3 position, the enzyme prefers Pro greater than Val greater than Leu greater than Ala greater than Gly, following the same order of preference seen in the tubulin cleavage pattern. Substitution of Leu for Ala in P1' and of Ser for Pro in P2' decreases activity by 10(5)- and 10(2)-fold, respectively. In position P3', substitution of Trp for Leu leaves the activity unaltered. However, introduction of the Trp fluorophore greatly enhances the sensitivity of the assay due to a 10-fold increase in indole fluorescence for cleavage of any peptide bond between the tryptophan and the dansyl group. Such an energy-transfer-based assay should have widespread use for detection of neutral proteases.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.

Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.

Amino Acids↗
PubMed1989

Biosynthesis of Astacus protease, a digestive enzyme from crayfish.

For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalloenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.

Animals↗
PubMed1987

Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic inflammatory bowel disease.

The sera of 59 patients with Crohn's disease (CD) and of 46 patients with ulcerative colitis (UC) were tested for autoantibodies (Aab) by indirect immunofluorescence with modern histochemical techniques using 19 different human tissues as antigenic substrates. Control collectives consisted of 19 patients with coeliac disease and of 100 healthy subjects. It was possible to demonstrate a specific marker for CD: Aab against exocrine pancreas (Pab) were present in 39% of the CD sera (UC 4%, coeliac disease 0%, healthy controls 3%). High Pab titres were only detectable in CD sera (29%). The CD-related autoantigen was demonstrated to be a component of normal pancreatic juice. Pab in CD were fundamentally different from those sometimes occurring in chronic and acute pancreatitis. It is suggested that CD is caused by autoimmune reactions against a component of pancreatic juice. Pab in CD correspond to Aab against intestinal goblet cells (Gab), which occurred exclusively in UC (28%). Pab and Gab, but obviously none of the other Aab investigated in this study, are of diagnostic value in chronic inflammatory bowel disease.

Adult↗
PubMed1984

[Autoantibodies against the exocrine pancreas and against intestinal goblet cells in the diagnosis of Crohn's disease and ulcerative colitis].

It was demonstrated by indirect immunofluorescence that Crohn's disease and ulcerative colitis are serologically distinct. In 59 patients with Crohn's disease, confirmed by endoscopy and histology, 23 (39%) had serum autoantibodies against exocrine pancreas; in 17 (29%) the titre was 1 : 100 or higher. In 46 patients with confirmed ulcerative colitis pancreas antibodies were demonstrated only twice, in 100 healthy control subjects only 3 times, with titres less than 1 : 100. Pancreas antibodies do not occur in high concentrations in pancreatitis; titres higher than 1 : 100 therefore suggest Crohn's disease. The pancreas antibodies of patients with Crohn's disease were predominantly immunoglobulins IgA and IgG, twice they were IgD and once IgM, never IgE. In 6 patients the pancreas antibodies fixed complement. Autoantibodies against intestinal goblet cells were found only in patients with ulcerative colitis (13 of 46 = 28%). The titres range was from 1 : 10 to 1 : 1000. The goblet-cell antibodies consisted only of IgA and IgG, never reacting with complement. These results indicate that determining pancreas and goblet-cell antibodies alone will make it possible to diagnose either Crohn's disease or ulcerative colitis in more than a quarter of patients with chronic inflammatory intestinal disease.

Adolescent↗