Robert Zwilling

Biosynthesis of Astacus protease, a digestive enzyme from crayfish

SummaryFor the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalfoenzyme 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.

Low molecular mass protease: evidence for a new family of proteolytic enzymes

We have isolated from decapode crustacea a proteolytic enzyme with the remarkably low M, value of 11 000 [I]. This low M, protease is synthesized in the hepatopancreas and secreted into the stomachlike cardia where it serves digestive processes. We have investigated the immunological properties of the low MI protease in the species Astucus jluviatilis, Astacus leptodactvlus (crayfish) as well as in Carcinus rnaenas (crab) [2,3]. The enzyme occurs in multiple and closely related forms varying from species to species [41. Proteolytic enzymes with similar properties have been noticed in other crustacea, e.g., Orconectes virilis [5], Homarus americanus [6] and Penaeus setiferus [7]. However, the distribution of low M, protease in other invertebrate species is still unknown, and in vertebrate animals this enzyme is apparently lacking completely. The amino acid composition, length of the protein chain, and other observations suggested that the lowM, protease might be a member of a hitherto unknown family of proteolytic enzymes [8]. This finding has been confirmed by the results of chemical modification, cleavage specificity toward peptide substrates, and partial amino-terminal sequence analysis of the enzyme.

α2-macroglobulin from the haemolymph of the freshwater crayfish Astacus astacus

Abstract 1. 1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate α2-macroglobulin. 4. 4. The α2-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. 4. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. 5. The N-terminal amino acid sequence of Astacusα2-macroglobulin is strongly related to the α2-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 α2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human α2-macroglobulin.

A protease from Astacus fluviatilis as an aid in protein sequencing

Abstract A protease from the digestive tract of the crayfish Astacus fluviatilis (EC 3.4.99.6) which does not seem to belong to any of the known families of proteases, proves to be a valuable aid in sequencing proteins. In denatured polypeptide chains, it has the unusual property of hydrolyzing peptide bonds on the amino side of the small uncharged residues Ala, Thr, Ser, Gly, and Val. The number of cleavage sites, however, is restricted to about one-fifth of the respective residues present. A requirement for cleavage may be the capability of the polypeptide chain to assume a conformation similar to a reverse turn at the site of hydrolysis.

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.

Kinetic evidence for cooperative binding of two orthophenanthroline 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.

Amino-terminal amino acid sequences and the evolution of frog (Rana esculenta) trypsin and chymotrypsin

At present it is unknown at what point during the molecular evolution of the serine proteases trypsin and chymotrypsin diverged from each other. Chymotrypsin has been found in all vertebrates that have been examined [ 11, whereas no chymotrypsin-like esterase activity is detectable in the digestive tract of the invertebrate crayfish, Asfacus jluviatilis [2]. Crayfish trypsin [3] as judged by its amino-terminal sequence and the number and position of the disulfide bonds, appears to have evolved before trypsin and chymotrypsin diverged from a common ancestor, suggesting that the divergence occurred sometime before the appearance of the vertebrate species. Since amphibians are much closer to the origin of vertebrate evolution and hence to the trypsin/chymotrypsin divergence than are many other vertebrate species, we examined amphibian trypsin and chymotrypsin, assuming that these enzymes in lower vertebrates resemble to a greater extent the common ancestral serine protease from which trypsin and chymotrypsin diverged than do bovine trypsin and chymotrypsin. Here we report the amino terminal sequences of trypsin and chymotrypsin from the frog Rarza esculen ta.