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Serine proteases of sea anemone Gibson, David McLaren

Abstract

A study of the proteolytic enzymes of the sea anemone Metridium senile was undertaken in search of the proposed archetype 'serine protease' from which the family of homologous serine proteases of higher animals is believed to have arisen. Activity towards the chymotrypsin specific substrate N-acetyl-L-tyrosine ethyl ester (ATEE) was found to be initially very low in homogenates of gastric filaments of Metridium but during incubation at 0°C an autocatalytic increase in the activity was observed. This 'activation' was inhibited by a competitive inhibitor of trypsin, benzamidine and by di-isopropylphosphorofluoridate (DFP) but not by the chymotrypsin competitive inhibitor indole. These observations suggested a process somewhat similar to the activation of serine proteases of higher animals. General steps in the purification of the anemone proteases were as follows: (i) 2 M LiCl extraction of acetone powder of gastric filaments (ii) acetone fractionation of the 2 M LiCl extract at -15°C, (iii) DEAE-Sephadex chromatography of the 40-80% acetone fraction, (iv) cation exchange chromatography of the DEAE-breakthrough peak on Bio-Rex 70 at pH 6.0 in the presence of .005 M indole and (v) re-chromatography on Bio-Rex 70 at pH 6.0 in the presence of indole. ATEE-ase activity was resolved into 3 components, designated A, BI and B2 in step (iv). Peaks A and BI migrated as single major Amido Black staining bands at pH 4.0 on polyacrylamide disc electrophoresis but there were several minor components present in both cases. Protease B2 appeared homogeneous on electrophoresis. 8 M urea-starch gel electrophoresis 32 of Dl³² P-labelled proteases followed by autoradiography; and Amido Black staining showed that the major Amido Black staining band was also the major radioactive band in each case. Gel filtration of protease A on G-100 Sephadex gave an approximate molecular weight of 40-50, 000. Sedimentation velocity of the enzymes gave values of S₂₀ of 2.0, 2.5. and 2.2 for DIP-proteases A, BI and B2, respectively, indicative of a lower molecular weight. Sedimentation equilibrium studies with protease A indicated considerable polydispersity but gave a weight-average molecular weight (Mw) of around 28, 000. The behavior on Sephadex G-100 and evidence of polydispersity upon sedimentation equilibrium may be a result of dimerization. Features of the amino acid composition showing similarity to α-chymotrypsin were a high half-cystine content and a similar number of aromatic and basic amino acids. Major differences from mammalian α-chymotrypsin included a higher proline and histidine content and a lower content of hydroxylic amino acids. Enzymic properties of the anemone enzymes were very similar to those of mammalian α-chymotrypsin. Protease A showed 3-5 fold higher k[subscript: cat] values in the hydrolysis of several chymotrypsin ester substrates but a markedly lower k[subscript: cat] in the hydrolysis of N-acetyl-L- tryptophan ethyl ester. The anemone enzyme was shown to be rapidly inhibited by DFP, phenyl methane sulphonyl fluoride (PMSF) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) but was not inhibited by the trypsin specific inhibitor N-[symbol omitted]tosyl-L-lysine chloromethylketone (TLCK). Competitive inhibition of the hydrolysis of ATEE was observed with indole and 3-phenyl propionate. Protease A hydrolysed the polypeptide chain of glucagon on the carboxyl side of tyrosine, phenylalanine and leucine, provided the latter was not adjacent to tyrosine or phenylalanine. A lower but significant cleavage at both sides of the two arginine residues was also observed. The latter cleavages were not affected by the chymotrypsin inhibitor TPCK but were eliminated by prior reaction of the enzyme with soybean trypsin inhibitor. Thus it was concluded that they represent the activity of a contaminating trypsin-like enzyme in the preparation of protease A. In studies on the short active centre sequence of proteased A, BI and B2 using the technique of co-electrophoresis of partial acid hydrolysates of Dl³² P-labelled enzymes and Dl³² P-chymotrypsin followed by high voltage electrophoresis and autoradiography, the active centre sequence of the three anemone proteases was found to be -Asp. Ser. Gly-, the sequence common to all of the mammalian serine proteases. In conclusion, the anemone enzymes show remarkable structural and enzymic similarity with mammalian chymotrypsin and they probably represent present day relics of early forms of chymotrypsin, homologous with the familiar enzyme of higher animals.

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