Molecular relationship between two types of phospholipase B from Penicillium notatum and reconstitution of active enzyme from its peptide fragments.
Two types of phospholipase B of Penicillium notatum, the native type and the modified type that is thought to be generated by the introduction of some nicks into the native type of enzyme by the endogenous protease(s), were distinguished on a slab sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under a nonreducing condition. The native form migrated with a rate corresponding to 95K Da, whereas the modified form migrated more slowly, corresponding to 106K Da, presumably because of its more extended conformation. That the "106K" protein was indeed a nicked product of the "95K" protein was confirmed by amino acid analysis, peptide mapping, N- and C-terminal sequence analyses, and immunoblotting. The peptide fragments (70K and 37K + 32K) comprising the modified protein were isolated by gel filtration in the presence of SDS and 2-mercaptoethanol (the 32K peptide was suggested to be a partial proteolytic product of the 37K peptide). When the "95K" protein was subjected to the same treatment under denaturing condition, it retained a low, but significant, enzymatic activity; in contrast, the separated peptide fragments did not show any significant activity. By a coincubation of these fragments, however, a restoration of enzymatic activity was observed through a reformation of the active complex, corresponding to the original modified protein. The enzymatic activity of this complex was further increased by a treatment with guanidine X HCl, followed by dialysis. The association of peptide fragments appears to occur through the formation of interpeptidal disulfide bonds.
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines; D008245:Lysophospholipase / Q000378:metabolism*; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D006146:Guanidines; D010446:Peptide Fragments; D010740:Phospholipases; D008245:Lysophospholipase; D010447:Peptide Hydrolases; D014357:Trypsin; D019791:Guanidine