The catalytic subunit of the cAMP-dependent protein kinase of ovine sperm flagella has a unique amino-terminal sequence.
The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated. Ram sperm C was purified and found by mass spectrometry (MS) to be approximately 890 Da smaller than Calpha, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Calpha, but differed from the predicted sequences for the Cbeta and Cgamma isoforms. MS analysis of 2-nitro-5-thiocyanatobenzoic acid fragments showed that the mass difference originated in the amino-terminal region. A unique blocked amino-terminal fragment was isolated from sperm C and sequenced by a combination of tandem mass spectrometry and Edman degradation of a subfragment. The results revealed that the amino-terminal myristate and the first 14 amino acids of Calpha are replaced by an amino-terminal acetate and six different amino acids in sperm C. The predicted mass difference due to these changes is 899 Da. The region of homology between sperm C and Calpha begins at the exon 1/exon 2 boundary in Calpha, suggesting that sperm C results from use of an alternate exon 1 in the Calpha gene. The different amino terminus of sperm C may be related to a unique requirement for localization of the "free" C subunit within the sperm flagellum.
San Agustin
J T
JT
Department of Cell Biology, University of Massachusetts Medical Center, Worcester Foundation Campus, Shrewsbury, Massachusetts 01545, USA.
Leszyk
J D
JD
Nuwaysir
L M
LM
Witman
G B
GB
D000085:Acetates / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D003488:Cyanogen Bromide; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D005091:Exons; D007438:Introns; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D019814:Myristic Acid / Q000032:analysis; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013077:Sperm Head / Q000201:enzymology; D013082:Sperm Tail / Q000201:enzymology*; D014357:Trypsin
D000085:Acetates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019814:Myristic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin; D003488:Cyanogen Bromide