Cloning and characterization of two alternatively spliced rat alpha-amidating enzyme cDNAs from rat medullary thyroid carcinoma.
The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.
Bertelsen
A H
AH
Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
Beaudry
G A
GA
Galella
E A
EA
Jones
B N
BN
Ray
M L
ML
Mehta
N M
NM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002277:Carcinoma / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D015183:Restriction Mapping; D013964:Thyroid Neoplasms / Q000201:enzymology / Q000235:genetics*; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D004247:DNA; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors