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Biomedical subjects

R Zwilling

Publications and source records attributed to R Zwilling.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1987

Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis.

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.

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↗
PubMed1983

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If.

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.

Amino Acid Sequence↗
PubMed1983

Enkephalin-degrading activity in Xenopus laevis tadpoles and adults blood.

The enkephalin-degrading enzyme (alpha-aminoacyl-peptide hydrolase,EC 3.4.11.11) responsible for the rapid inactivation of enkephalins in human blood was purified and compared to the enkephalin-degrading system of Xenopus laevis tadpoles and adult blood. The specificity and the kinetic constants of the Xenopus enzyme(s) were determined after partial purification. Even if remarkable similarities between the Xenopus and the human enzyme exist, still they show differences in specificity towards peptides whose N-terminal is Phe. Amphibians are able to manufacture enkephalins and the present work shows that they are endowed with an enkephalin-degrading system comparable to the soluble one of human blood.

Amino Acid Sequence↗
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↗