ALKALINE INORGANIC PYROPHOSPHATASE FROM MICROCYSTIS AERUGINOSA

Abstract

The strength of binding of horse-heart cytochrome c, myoglobin and chymotrypsinogen A to Sephacryl S-200 increased with increasing temperature and increasing ammonium sulphate concentrations. These proteins could be displaced by lowering the salt concentration of, or by the addition of glycerol to the eluent buffer. The proteins were eluted in the order of increasing hydrophobicities -- cytochrome c, myoglobin and finally, chymotrypsinogen A. These observations presented evidences that the interaction between the proteins and the Sephacryl gel matrix was probably of a hydrophobic nature. The pH of the eluent buffer modified the binding capacity of the individual proteins by altering their surface charges. At or near its pH, the ability of a protein to interact in a hydrophobic manner was enhanced and it would therefore bind more tightly to the gel. This was successfully demonstrated using the protein myoglobin. Optimal resolution of cytochrome c, myoglobin and chymotrypsinogen A on Sephacryl S-200 was achieved- with flow rates up to 0.48 ml min-1. Flow rates higher than this did not cause a collapse or shrinkage of the Sephacryl column but resolution of the proteins was lost. Sephacryl S-1000 proved to be the best adsorbent. The three proteins were also observed to be best-resolved when chromatographed on a Sephacryl S-1000 column. Fractionation of crude Microcystis aeruginosa extract on a Sephacryl S-200 column (equilibrated with 60% ammonium sulphate-saturated buffer) resulted in the concentration of alkaline PPase and more importantly, in the complete removal of contaminating phycobiliproteins from the enzyme. Fractions containing alkaline PPase were pooled and further subjected to ion-exchange chromatography and gel filtration. The final preparation of the enzyme was judged homogeneous by electrophoresis on polyacrylamide gels of different concentrations as well as by electrophoresis in the presence of sodium dodecylsulphate. Homogeneity was further confirmed by N-terminal amino acid analysis. Alkaline PPase from M. aeruginose required Mg 2+ for its enzymatic activity. Maximal hydrolysis of PPi was obtained at a Mg 2+:PPi ratio of 10: 1 and at pH 7.5. Maintaining Mg2+ at 10 mM and pH at 7. 5, Km and Vmax were determined to be 1. 33 mM PPi and 3.33 mM Pi min-1mg-1 protein respectively. The molecular weight of M. aeruginosa alkaline PPase was 80,000 to 87,000 daltons as determined by disc gel electrophoresis and gel filtration. Results from SDS gel electrophoresis showed that it is a trimer, with subunits of about 28,000 daltons each. Isoelectric focusing on polyacrylamide gels of M. aeruginosa alkaline PPase showed numerous bands, which did not reflect its homogeneity. A minor protein band at pH 4.44 and multiple bands at pH ranging from 7.35 to 7. 69 were observed. The pH of the enzyme, however, was confirmed to be 4.45 by chromatofocusing. Alkaline PPase from M. aeruginosa was extremely rich in asparagine and aspartic acid but poor in cysteine, serine and methionine. Glutamine, glutamic acid, lysine and leucine were also present in considerable numbers. A sequence of forty-three amino acid residues, although the identity of a few were indefinite, was determined from the N-terminal of M. aeruginosa alkaline PPase. No region of homology was observed when this sequence was compared to that obtained for yeast or Escherichia coli alkaline PPase. Antibody against homogeneous M. aeruginosa alkaline PPase has been successfully generated in chickens.