title: Precursor-product relationship of larger to smaller molecular forms of the BAL 31 nuclease from Alteromonas espejiana: preferential removal of duplex exonuclease relative to endonuclease activity by proteolysis.
abstract: Two molecularly and kinetically distinct major species of the extracellular nuclease BAL 31 from Alteromonas espejiana, previously characterized as the "fast" (F) and "slow" (S) BAL 31 nucleases, have been evidenced to derive from proteolysis starting from a still larger (approximately 120 kDa) precursor nuclease. The expected protease activity in the culture fluid has been confirmed and is strongly dependent on the cell growth phase. The disappearance of the largest nuclease species with the concomitant sequential appearance of first the F and then the S species has been demonstrated for nuclease obtained from culture supernatants as a function of cell growth phase. Nuclease from periplasmic extracts displayed very little of the F and S nucleases. Treatment of purified F nuclease with Pronase or subtilisin readily converted it to species with only a few percent of the native exonuclease activity against duplex DNA but retaining much of the initial activity against single-stranded DNA. Electrophoresis in nuclease-detecting gels demonstrated a parallel conversion of the larger species to one indistinguishable in molecular weight from the S species. The observed loss of exonuclease activity could correspond to the conversion of the F to the S nuclease. However, treatment of S nuclease with subtilisin resulted in a drastic reduction of exonuclease activity of this enzyme on duplex DNA with retention of most of the activity against single-stranded and nicked circular duplex DNA substrates. Evidence of internal proteolysis of the S nuclease could be seen after electrophoresis in denaturing gels but only after the denaturation buffer was adjusted to 6 M in urea. The preferential removal of the exonuclease activity may enhance the usefulness of the BAL 31 nuclease in such applications as heteroduplex mapping.
authors.affiliation: Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5500.
authors.forename: C R
authors.initials: CR
authors.lastname: Hauser
authors.forename: H B
authors.initials: HB
authors.lastname: Gray
mesh_terms: D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004792:Enzyme Precursors / Q000302:isolation & purification / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D010957:Plasmids
chemical_list: D004792:Enzyme Precursors; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31