The Catalytic Subunit of the cAMP-dependent Protein Kinase of Ovine Sperm Flagella Has a Unique Amino-terminal Sequence*
- From the ‡Department of Cell Biology and§Protein Chemistry Facility, University of Massachusetts Medical Center, Worcester Foundation Campus, Shrewsbury, Massachusetts 01545 and ¶Perkin Elmer Sciex, Applied Biosystems Division, Foster City, California 94404
Abstract
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 ∼890 Da smaller than Cα, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Cα, but differed from the predicted sequences for the Cβ and Cγ 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 Cα 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 Cα begins at the exon 1/exon 2 boundary in Cα, suggesting that sperm C results from use of an alternate exon 1 in the Cα 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.
Footnotes
-
↵* This study was supported by National Institutes of Health Grant HD23858, National Science Foundation Grant 9512226, and a grant from the Campbell and Hall Charity Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵2 J. T. San Agustin and G. B. Witman, unpublished results.
- Abbreviations:
- PKA
-
cAMP-dependent protein kinase
- C
-
catalytic subunit of PKA
- R
-
regulatory subunit of PKA
- PKI
-
heat stable inhibitor of PKA
- CNBr
-
cyanogen bromide
- NTCB
-
2-nitro-5-thiocyanatobenzoic acid
- PAGE
-
polyacrylamide gel electrophoresis
- PVDF
-
polyvinylidine difluoride
- DTT
-
dithiothreitol
- E-64
-
trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane
- TLCK
-
N α-p-tosyl-l-lysine chloromethyl ketone
- TPCK
-
N-tosyl-l-phenylalanine chloromethyl ketone
- MALDI TOF MS
-
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- HBTU
-
o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate
- Tricine
-
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine
- HPLC
-
high performance liquid chromatography.
-
- Received June 19, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
