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Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium
D A Stoffers1, C B Green, B A Eipper
1Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Abstract:
Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions.
Insights
Peptidyl-glycine alpha-amidating monooxygenase (PAM) is crucial for creating active peptides. Alternative splicing creates two PAM mRNA forms in rat hearts, differing by a 315-base-pair segment, impacting protein processing.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Peptidyl-glycine alpha-amidating monooxygenase (PAM) is essential for post-translational modification of peptides.
- PAM catalyzes the amidation of glycine-extended peptides, a critical step for biological activity.
- This enzymatic activity requires copper, ascorbate, and molecular oxygen.
Purpose of the Study:
- To isolate and sequence cDNAs for the two major PAM mRNA classes in adult rat heart atrium.
- To investigate the structural differences and origins of these mRNA variants.
- To analyze the conservation of PAM across species.
Main Methods:
- cDNA isolation and sequencing from adult rat heart atrium.
- Northern blot analysis to detect and differentiate PAM mRNA forms.
- Nucleotide sequence comparison across rat, frog, and bovine PAM cDNAs.
Main Results:
- Two distinct cDNA types, rPAM-1 and rPAM-2, were identified, differing by a 315-bp deletion in rPAM-2.
- Northern analysis confirmed that the larger PAM mRNA form, corresponding to rPAM-1, is present in adult rat atrium.
- The deleted segment encodes a 105-amino acid region with N-glycosylation and potential endoproteolytic processing sites.
- Highly conserved nucleotide sequences in the 3' untranslated region and amino acid sequences across species were observed.
Conclusions:
- The two PAM mRNA forms in rat atrium likely arise from alternative splicing.
- The presence or absence of the 315-nucleotide segment significantly distinguishes the mRNA forms.
- Conserved domains suggest critical roles for both catalytic and non-catalytic regions of PAM in various species.