Clarification of an Uncertain Intron within the cDNA Sequences of Arrowhead Proteinase Inhibitors A and B

• Published in: Journal of biochemistry (Tokyo) 1997-05, Vol.121 (5), p.991-995
• Main Authors: Luo, Ming-Juan, Lu, Wu-Yuan, Chi, Cheng-Wu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequence determination; arrowhead proteinase inhibitor; Base Sequence; DNA, Complementary - chemistry; DNA, Complementary - genetics; Introns - genetics; Molecular Sequence Data; mutation; Plant Proteins - chemistry; Plant Proteins - genetics; Protease Inhibitors - chemistry; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; sitedirected
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a021685

An uncertain intron of 87 bp within the cDNA sequences of arrowhead proteinase inhibitors Aand B was clarified. By site-directed mutation with either a stop codon inside the uncertain intron or mutated codons at both its 5′ and 3′ ends, it was proved that there was neither a translation intron nor a protein intron present in the cDNA sequences of proteinase inhibitors A and B. The primary structure of inhibitor B was then reexamined by mass spectrometry molecular weight determination and partial amino acid sequencing. A 38 residue peptide was derived by degradation of inhibitor B with lysylendopeptidase, and purified, which was not found in the previous work, and its N-terminal part was none other than the missed 29 residue peptide encoded by the uncertain intron. The 38 residue peptide was very hydrophobic, while the 29residue peptide it included was even more hydrophobic. The N-terminal part of the missedpeptide was also aligned within a BrCN-degraded fragment of the inhibitor. In this paper the cause of the overlooking of this 29 residue peptide in the previous work and some unexpected problems which arose during the former sequence analysis are explained.