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P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species.
Abstract: P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.
Publication Date: 1994-07-01 PubMed ID: 8033890DOI: 10.1111/j.1432-1033.1994.tb18979.xGoogle Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research explores the phosphorylation reactions of P1 and P2 protamines using different protein kinases in five mammalian species – human, stallion, bull, boar, and ram. This experiment finds protein kinase A (PKA) is used to phosphorylate P1 protamines while protein kinase C (PKA) is the preferred kinase for P2 protamines, suggesting there are other kinds of protamine kinases in sperm cells responsible for their phosphorylation.
Methodology and Findings
- The researchers took P1 protamines from the spermatozoa of human, stallion, bull, boar and ram, and P2 protamines from the spermatozoa of human and stallion.
- These were then subjected to phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC).
- PKA phosphorylated all P1 protamines while PKC only phosphorylated P2 protamines.
- Added to this, human, stallion and boar P1 protamines but not bull and ram, were also phosphorylated by PKC.
Phosphoamino Acid Analysis
- Following phosphoamino acid analysis, protamines with positive indications for phosphoserine (P-Ser) were subjected to P-Ser conversion reactions and protein sequencing.
- Stallion and human P1 protamines had P-Ser present post-PKA phosphorylation which was situated in the middle region of the molecule at specific points in each species.
- All other phosphorylated P1 protamines contained only P-Thr, which couldn’t be localized in the sequence with the methods used.
- Post-PKC phosphorylation, the internally located Ser residues in human and stallion P1 protamines were phosphorylated and a specific site was slightly phosphorylated in boar P1 protamine.
Additional Observations
- The researchers noted that Ser residues in P1 protamines known to be phosphorylated in vivo were not phosphorylated by either kinase used in this study.
- This indicates that other types of protamine kinases present in sperm cells are responsible for their phosphorylation.
- Within P2 protamines, one type in humans was equally phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas in stallions, a specific site was the main target for PKC phosphorylation in vitro.
Conclusion
- The research concludes that PKC is a good candidate for in vivo phosphorylation of P2 protamines while PKA might be used for phosphorylating some hydroxyamino acid residues in P1 protamines.
- This suggests a distinct preference of kinase use for the different types of protamines, indicating differing phosphorylation processes between P1 and P2 protamines.
Cite This Article
APA
Pirhonen A, Linnala-Kankkunen A, Mënpää PH.
(1994).
P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species.
Eur J Biochem, 223(1), 165-169.
https://doi.org/10.1111/j.1432-1033.1994.tb18979.x Publication
Researcher Affiliations
- Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
MeSH Terms
- Amino Acid Sequence
- Animals
- Cattle
- Cyclic AMP-Dependent Protein Kinases / metabolism
- Horses
- Humans
- Male
- Molecular Sequence Data
- Phosphorylation
- Protamines / metabolism
- Protein Kinase C / metabolism
- Sequence Homology, Amino Acid
- Sheep
- Species Specificity
- Spermatozoa / metabolism
- Swine
Citations
This article has been cited 4 times.- Moritz L, Schon SB, Rabbani M, Sheng Y, Agrawal R, Glass-Klaiber J, Sultan C, Camarillo JM, Clements J, Baldwin MR, Diehl AG, Boyle AP, O'Brien PJ, Ragunathan K, Hu YC, Kelleher NL, Nandakumar J, Li JZ, Orwig KE, Redding S, Hammoud SS. Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1. Nat Struct Mol Biol 2023 Aug;30(8):1077-1091.
- Gou LT, Lim DH, Ma W, Aubol BE, Hao Y, Wang X, Zhao J, Liang Z, Shao C, Zhang X, Meng F, Li H, Zhang X, Xu R, Li D, Rosenfeld MG, Mellon PL, Adams JA, Liu MF, Fu XD. Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation. Cell 2020 Mar 19;180(6):1212-1227.e14.
- Rose KL, Li A, Zalenskaya I, Zhang Y, Unni E, Hodgson KC, Yu Y, Shabanowitz J, Meistrich ML, Hunt DF, Ausió J. C-terminal phosphorylation of murine testis-specific histone H1t in elongating spermatids. J Proteome Res 2008 Sep;7(9):4070-8.
- Burton KA, McDermott DA, Wilkes D, Poulsen MN, Nolan MA, Goldstein M, Basson CT, McKnight GS. Haploinsufficiency at the protein kinase A RI alpha gene locus leads to fertility defects in male mice and men. Mol Endocrinol 2006 Oct;20(10):2504-13.
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