Pirhonen A, Linnala-Kankkunen A, Mënpää P H
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Eur J Biochem. 1994 Jul 1;223(1):165-9. doi: 10.1111/j.1432-1033.1994.tb18979.x.
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.
从人、种马、公牛、公猪和公羊的射出精子中分离出的P1鱼精蛋白以及来自人和种马精子的P2鱼精蛋白，在碱性磷酸酶处理后，使用环磷酸腺苷依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）进行体外磷酸化反应。所有P1鱼精蛋白都被PKA磷酸化，而P2鱼精蛋白仅被PKC磷酸化。此外，人、种马和公猪的P1鱼精蛋白（但公牛和公羊的P1鱼精蛋白未被磷酸化）被PKC磷酸化。进行磷酸氨基酸分析后，对显示磷酸丝氨酸（P-Ser）阳性信号的鱼精蛋白进行P-Ser转化反应和蛋白质测序。PKA磷酸化后，只有种马（St1）和人（HP1）的P1鱼精蛋白含有P-Ser，位于分子的中间区域，即St1中的Ser29和HP1中的Ser28。所有其他磷酸化的P1鱼精蛋白仅含有P-Thr，用目前的方法无法在序列中进一步定位。PKC磷酸化后，人（ser21）和种马（Ser29）P1鱼精蛋白内部的Ser残基被磷酸化，在公猪P1鱼精蛋白中，只有Thr43被轻微磷酸化。P1鱼精蛋白中已知在体内被磷酸化的N端Ser残基，未被任何一种激酶磷酸化，这表明精子细胞中一定还有其他类型的鱼精蛋白激酶负责它们的磷酸化。在P2鱼精蛋白中，除了一些Thr磷酸化外，HP2在所有Ser残基上的磷酸化程度相同，而在St2中，Ser32是体外PKC磷酸化的主要靶点。总的来说，PKC是P2鱼精蛋白体内磷酸化的良好候选者，而PKA是P1鱼精蛋白中一些羟基氨基酸残基磷酸化的候选者。
