Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA.
Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase A1. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Methods Enzymol. 197, 446-456], Penicillium notatum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged. A cDNA library of P. notatum was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization-positive clone, pPLB18, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA library with a Sau3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues. From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N-glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases.
Masuda
N
N
Department of Medical Chemistry, Kansai Medical University, Osaka, Japan.
Kitamura
N
N
Saito
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D004271:DNA, Fungal / Q000235:genetics; D015723:Gene Library; D008245:Lysophospholipase / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D008245:Lysophospholipase