The minimal functional sequence of protamine

Despite its nearly universal applications, protamine, a mixture of four major peptides with different sequences, is associated with clinically significant side effects. Through a well-designed enzyme digestion method, various low molecular weight protamine peptides were obtained. Among them, two low molecular weight protamine peptides with the same or even more potent heparin neutralization abilities as native protamine were identified through both in vitro and in vivo tests. In addition, in vivo experiments showed that compared to native protamine, these two low molecular weight protamine peptides were less toxic and would be safer for clinical use.     

Studies of the injection of poly(A) protamine mRNA into Xenopus laevis oocytes

When poly(A)⁺ protamine mRNA from trout testes polysomes was injected into living Xenopus oocytes and the latter labelled with [¹⁴C] or [³H]arginine during subsequent incubation, a highly basic, labelled protein fraction was synthesized and could be extracted with 0.5 M H₂SO₄. In the acid extract, a major polypeptide, indistinguishable from trout protamine by several criteria: polyacrylamide and starch gel electrophoreses, carboxymethylcellulose column chromatography, lack of incorporation of [³H]histidine, and autoradiography of tryptic peptides after two-dimensional paper electrophoresis, could be demonstrated. Since no such protein is found in control oocytes injected with saline, it is concluded that poly(A)⁺ protamine mRNA programs the synthesis of trout protamine within Xenopus oocytes. This confirms our previous reports [1–3] that trout testis poly(A)⁺ protamine mRNA can direct the in vitro synthesis of protamine in Krebs II ascites, rabbit reticulocytes and wheat germ cell-free systems. The protamine synthesized upon injection of poly(A)⁺ protamine mRNA into Xenopus oocytes appears to be partially phosphorylated. Injection of increasing amounts of poly(A)⁺ protamine mRNA led to a linear increase in protamine synthesis. The sensitivity of detection was such that less than 1 ng of poly(A)⁺ protamine mRNA gave a significant response. The translational stability of protamine mRNA appeared to be less than that of globin mRNA.     

Zinc is sufficiently abundant within mammalian sperm nuclei to bind stoichiometrically with protamine 2

Although studies have demonstrated that zinc can bind to sperm nuclear proteins, specifically protamine 2, it has not been shown that the metal is sufficiently abundant inside the sperm nucleus to interact stoichiometrically with these proteins. In this study proton-induced X-ray emission (PIXE) has been used to measure the amount of sulfur and zinc within the nuclei of individual sperm cells to infer the stoichiometry of zinc binding to protamine 2 in six species of mammal: bull, chinchilla, stallion, hamster, human, and mouse (protamine 2 comprises from 0% (bull) to 67% (mouse) of the protamine present in the sperm of these animals). Using the sulfur mass and electrophoretic data on the relative proportion of protamine 1 and protamine 2 in the sperm chromatin of these species, the protamine 1, protamine 2, and total protamine contents within each species sperm nuclei have been determined. The PIXE measurements reveal that the zinc content of the sperm nucleus varies proportionately with the protamine 2 content of sperm chromatin. PIXE analyses of hamster protamines extracted under conditions that appear to at least partially preserve zinc binding also confirm that the majority of the metal is bound to protamine. In five of the species examined, sufficient zinc is present for each protamine 2 molecule to bind one zinc. The results obtained for chinchilla sperm, conversely, indicate the chinchilla protamine 2 molecule may interact differently with zinc. Chinchilla sperm only contain enough zinc for one atom to be bound to two protamine 2 molecules.     

鱼精蛋白抗菌机制的研究

鱼精蛋白是一种存在于各类动物精巢组织中的多聚阳离子肽,其抗菌性很早就被所知,然而它的抗菌机理却一直未能得到很清楚的了解。现存在的机理有2种:一种认为鱼精蛋白与细菌细胞壁结合,通过破坏细胞壁的形成来达到抑菌效果;另一种认为鱼精蛋白破坏了细胞能量的转换、营养物质的吸收功能,细胞质膜是鱼精蛋白攻击的对象。事实上,作者认为,鱼精蛋白的抗菌效果可能是通过以上2种方式共同作用的结果,因而它的抗菌机理也可能是这两种机理的叠加,这还需进一步的研究证明。     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

A less toxic heparin antagonist--low molecular weight protamine

A new thirteen amino acid peptide, named low molecular weight protamine (LMWP), was obtained through the enzymatic digestion of native protamine. Both in vitro and in vivo results showed that LMWP fully maintained the heparin neutralization function of protamine but had much lower immunogenicity and antigenicity. Unlike protamine, neither LMWP nor LMWP/heparin complexes caused significant blood platelet aggregation in rats. These results suggest that LMWP can be used as a substitute for protamine for developing a new generation of nontoxic heparin antagonists.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.     

The lack of protamine 2 (P2) in boar and bull spermatozoa is due to mutations within the P2 gene.

The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.     

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.     

Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.     

Protamine immobilization and heparin adsorption on the protamine-bound cellulose fiber membrane

Immobilization of protamine to the inner lumen of cellulose hollow fibers has been shown useful in preventing both heparin- and protamine-induced complications during an extracorporeal blood circulation procedure. The current study examined the effects of variables on the immobilization of protamine to cyanogen bromide (CNBr)-activated cellulose hollow fibers. The degree of protamine immobilization was controlled by three independent parameters: the amount of CNBr used during the activation process, the duration of the coupling process, and the protamine concentration in the coupling solution. By the adjustment of these parameters, cellulose fibers containing desired amounts of immobilized protamine (ranging from 1 to 20 mg of immobilized protamine per gram of dry fibers) were readily prepared.Heparin adsorption to the protamine-bound cellulose fibers was also examined. The adsorption isotherm followed a Langmuir adsorption model. The amount of heparin adsorbed was dependent on both the heparin concentration in the substrate solution and the protamine loading on the fibers. The Langmuir adsorption constant K was estimated to be 0.37 +/- 0.06 mL/mg, whereas the saturation capacity Q(s) of the protamine-bound fibers increased with increasing the protamine loading.     

A rapid method for separating small vesicles from suspension.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.     

A novel label-free upconversion fluorescence resonance energy transfer-nanosensor for ultrasensitive detection of protamine and heparin

A novel label-free fluorescence nanosensor was developed for ultrasensitive detection of protamine and heparin based on fluorescence resonance energy transfer (FRET) between NaYF₄:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The FRET system was formed by the electrostatic adsorption of AuNPs on UCNPs, and the fluorescence of UCNPs was significantly quenched. When protamine was added to the mixture of UCNPs–AuNPs, the AuNPs interacted with protamine and then desorbed from the surface of UCNPs and aggregated, resulting in the recovery of the fluorescence of UCNPs. On the addition of both protamine and heparin, the FRET system formed owing to the stronger interaction between heparin and protamine than that with AuNPs, leading to a marked fluorescence quenching of UCNPs. The concentrations of protamine and heparin were proportional to the changes of the fluorescence of UCNPs. The linear response range was obtained over the concentration ranges of 0.02 to 1.2 μg/ml and 0.002 to 2.0 μg/ml with low detection limits of 6.7 and 0.7 ng/ml for protamine and heparin, respectively. Simultaneous measurement of protamine and heparin in human serum can be achieved, suggesting that the nanosensor can be used in a complex biological sample matrix.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei.

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei

Summary Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts.Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days.The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.     

Mapping and Measuring DNA to Protein Ratios in Mammalian Sperm Head by XANES Imaging

We have used XANES imaging, which combines X-ray absorption near edge spectral features (XANES) with 50-nm-resolution X-ray microscopy, to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined; these species were chosen for analysis because their sperm contain marked differences in their protamine 1 and protamine 2 contents. The data we've obtained for bull, stallion, hamster, and mouse sperm suggest that the total nuclear protein to DNA ratio is similar in the sperm of many eutherian mammals. Since protamine constitutes the majority of the sperm nuclear protein, these results indicate that the total protamine content of sperm chromatin must be constant among mammalian species, independent of the extent of expression of the protamine 2 gene.