PubMed Health⌕ Search

Biomedical subjects

R Zwilling

Publications and source records attributed to R Zwilling.

12 recordsLinked to original sources
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↗
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

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↗
PubMed1989

Kinetic evidence for cooperative binding of two ortho-phenanthroline molecules to astacus protease during metal removal.

Kinetic evidence is presented that introduces a new possibility for a mechanism of metal removal from a protein by a chelator. Astacus protease is a 22,614 dalton zinc-metalloendopeptidase from the digestive tract of the freshwater crayfish. Recent studies have shown that it contains a single zinc atom and that removal of this metal yields inactive apo-enzyme, which can be reactivated upon readdition of zinc, cobalt, or copper. The enzyme is inactivated by metal chelators in a time and concentration dependent manner. The inactivation of Zn-Astacus protease by 1,10-phenanthroline (OP) can be monitored continuously in the presence of substrate. The concentration of substrate was found to have no effect on the inactivation rate, indicating that the chelator binding during inactivation is of the noncompetitive type. First-order rate constants for the inactivation process are seen to depend on the concentration of chelator in a sigmoidal manner. Based on mathematics analogous to that for cooperativity in enzyme-substrate kinetics, the deduction is made that there are two OP binding sites on the protein and that the rate of inactivation is related to the saturation of both sites with ligand. If one uses this model, the limiting rate constant of inactivation upon saturation of both sites with ligand is 6.76 x 10(-3) sec-1, and the half maximal rate occurs at an OP concentration of 6.52 mM. A mechanism is proposed wherein both protein bound chelators can cooperate during metal removal either by direct chelation of the metal or by allosteric means. The proposed model and the noncompetitive binding of chelator and substrate are discussed in relation to a recently proposed metal binding site.

Animals↗
PubMed1989

Protease inhibitors from Ecballium elaterium seeds.

Several protease inhibitors were found in the seeds of a Cucurbitacea, Ecballium elaterium, and were separated from one another by affinity and molecular sieve chromatography. Three main trypsin isoinhibitors were purified by ion-exchange chromatography and the sequence of the major one, EETI II, was elucidated and compared with other inhibitors of the squash family. It is a peptide of M.W. 3020 of strong inhibitory activity (Ka = 8 x 10(11) M-1) against trypsin, showing high Gly content, six half-cystine residues, but devoid of histidine, threonine, tryptophan, and tyrosine residues.

Amino Acid Sequence↗
PubMed1985

Enkephalin-degrading activity in arthropode hemolymphe.

Enkephalin and related peptides are rapidly inactivated in Astacus fluviatilis and Limulus polyphemus hemolymphe. At least three different enzymes, an aminopeptidase, a carboxypeptidase and a peptidyl-dipeptidase, acting concomitantly on the peptide substrates have been identified. The properties of these enzymes were characterized and they were compared to similar enzymes of the vertebrate blood. The opioid peptides appear to be extremely short lived in invertebrate hemolymphe, which presumably is a metabolic barrier to the action of active peptides stronger than vertebrate blood.

Amino Acids↗
PubMed1979

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.

Amino Acid Sequence↗
PubMed1976

[Quantitative structure comparison of protein surfaces by micro-complement fixation].

Microcomplement fication (MCF) is a rapid and most sensitive method for the quantitation of slight modifications on protein surfaces not accessible by other immunologic means. Single amino acid substitutions (0.7 percent sequence differences) are readily detected. A firm relationship between sequence information and immunologic distance as measured by MCF could be established. The rate of molecular evolution of homology proteins can be derived from MCF measurements without prior knowledge of primary structures.

Amino Acids↗
PubMed1975

[Accumulation of basic amino acid residues, rate of autolysis and relative length of gut during trypsin evolution (author's transl)].

The study of six trypsins (crayfish, dogfish, horse, pig, sheep, bovine) showed that among these the highest lysine + arginine content and the highest inactivation rate occur in specialized herbivorous animals with extreme length of gut. Since the autocatalytic degradation in trypsins can take place only at lysine or arginine residues, an accumulation of basic amino acids, the increase of the autocatalytic inactivation rate and the relative length of gut might be correlated during the evolution of mammalian trypsins.

Amino Acids↗