Arginine-rich Protamine Research Articles

Protamines and the sperm nuclear basic proteins Pandora's Box of insects.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

Assessment of the Neuroprotective Effects of Arginine-Rich Protamine Peptides, Poly-Arginine Peptides (R12-Cyclic, R22) and Arginine-Tryptophan-Containing Peptides Following In Vitro Excitotoxicity and/or Permanent Middle Cerebral Artery Occlusion in Rats.

We have demonstrated that arginine-rich and poly-arginine peptides possess potent neuroprotective properties with arginine content and peptide positive charge being particularly critical for neuroprotective efficacy. In addition, the presence of other amino acids within arginine-rich peptides, as well as chemical modifications, peptide length and cell-penetrating properties also influence the level of neuroprotection. Against this background, we have examined the neuroprotective efficacy of arginine-rich protamine peptides, a cyclic (R12-c) poly-arginine peptide and a R22 poly-arginine peptide, as well as arginine peptides containing tryptophan or other amino acids (phenylalanine, tyrosine, glycine or leucine) in in vitro glutamic acid excitotoxicity and in vivo rat permanent middle cerebral artery occlusion models of stroke. In vitro studies demonstrated that protamine and poly-arginine peptides (R12-c, R22) were neuroprotective. Arginine-tryptophan-containing peptides were highly neuroprotective, with R12W8a being the most potent arginine-rich peptide identified in our laboratory. Peptides containing phenylalanine or tyrosine substituted in place of tryptophan in R12W8a were also highly neuroprotective, whereas leucine, and in particular glycine substitutions, decreased peptide efficacy. In vivo studies with protamine administered intravenously at 1000nmol/kg 30min after MCAO significantly reduced infarct volume and cerebral oedema by 22.5 and 38.6%, respectively. The R12W8a peptide was highly toxic when administered intravenously at 300 or 100nmol/kg and ineffective at reducing infarct volume when administered at 30nmol/kg 30min after MCAO, unlike R18 (30nmol/kg), which significantly reduced infarct volume by 20.4%. However, both R12W8a and R18 significantly reduced cerebral oedema by 19.8 and 42.2%, respectively. Protamine, R12W8a and R18 also reduced neuronal glutamic acid-induced calcium influx. These findings further highlight the neuroprotective properties of arginine-rich peptides and support the view that they represent a new class of neuroprotective agent.

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

Optimizing Tethered Particle Motion to Measure DNA Compaction by Protamine

During mammalian spermiogenesis, there is a remarkable change in the nuclear morphology whereby the chromatin is completely remodeled. Specifically, the histone proteins that wrap the DNA into nucleosomes are removed, and the DNA is coated with a series of small (∼100-amino-acid), arginine-rich protamines that dramatically compact the DNA into a series of toroids. This replacement and compaction 1) reduces the head size of the sperm to enhance their hydrodynamicity, 2) decreases the likelihood of DNA damage, and 3) removes the epigenetic markers passed on through histone modifications. Here, we directly observe the steps of toroid formation and the underlying mechanics. We use a single molecule biophysics assay where we tether a particle (1-µm-diameter bead) to the DNA and to the sample surface. During DNA folding, the end-to-end length of the DNA decreases, decreasing the motion of the particle. This tethered particle motion (TPM) assay is useful because we do not apply any external forces, allowing us to measure reversible folding events. Previously, we saw ∼200 nm changes in length that suggest that the toroid forms by successive loops. Here, we optimized the assay to enhance efficiency and resolution by improving bead monodispersion, protein binding to the surface, and DNA purification.

Comparative structure of vertebrate sperm chromatin

A consistent feature of sperm nuclei is its exceptionally compact state in comparison with somatic nuclei. Here, we have examined the structural organization of sperm chromatin from representatives of three vertebrate lineages, bony fish (Danio rerio), birds (Gallus gallus domesticus) and mammals (Mus musculus) using light and transmission electron microscopy (TEM). Although the three sperm nuclei are all highly compact, they differ in morphology and in the complement of compaction-inducing proteins. Whereas zebrafish sperm retain somatic histones and a nucleosomal organization, in the rooster and mouse, histones are largely replaced by small, arginine-rich protamines. In contrast to the mouse, the rooster protamine contains no cysteine residues and lacks the potential stabilizing effects of S-S bonds. Protamine driven chromatin compaction results in a stable, highly condensed chromatin, markedly different from the somatic nucleosome-based beads-on-a-string architecture, but its structure remains poorly understood. When prepared gently for whole mount TEM, the rooster and mouse sperm chromatin reveal striking rod-like units 40-50 nm in width. Also present in the mouse, which has very flattened sperm nuclei, but not rooster, where nuclei take the form of elongated cylinders, are toroidal shaped structures, with an external diameter of about 90 nm. In contrast, similarly prepared zebrafish sperm exhibit nucleosomal chromatin. We also examined the early stages in the binding of salmine (the salmon protamine) to defined sequence DNA. These images suggest an initial side-by-side binding of linear DNA-protamine complexes leading to the nucleation of thin, flexible rods with the potential to bend, allowing the ends to come into contact and fuse to form toroidal structures. We discuss the relationship between these in vitro observations and the rods and toroids seen in nuclei, and suggest an explanation for the apparent absence of these structures in TEM images of fully condensed sperm nuclei.

MP66-11 NUCLEOSOME RETENTION IN SPERM-CHROMATIN: A RANDOM PROCESS OR AN ORGANIZED EVENT WITH A BIOLOGICAL FUNCTION?

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-11 NUCLEOSOME RETENTION IN SPERM-CHROMATIN: A RANDOM PROCESS OR AN ORGANIZED EVENT WITH A BIOLOGICAL FUNCTION? Birgit Samans, Yang Yang, Stefan Krebs, Helmut Blum, Klaus Steger, Wolfgang Weidner, Temuujin Dansranjavin, and Undraga Schagdarsurengin Birgit SamansBirgit Samans More articles by this author , Yang YangYang Yang More articles by this author , Stefan KrebsStefan Krebs More articles by this author , Helmut BlumHelmut Blum More articles by this author , Klaus StegerKlaus Steger More articles by this author , Wolfgang WeidnerWolfgang Weidner More articles by this author , Temuujin DansranjavinTemuujin Dansranjavin More articles by this author , and Undraga SchagdarsurenginUndraga Schagdarsurengin More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2058AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The hallmark of the last step of spermatogenesis, spermiogenesis, is the condensation of paternal chromatin in order to generate a fertile sperm with a protected and transportable DNA. This is achieved by exchange of nucleosomes by small arginine-rich protamines. Interestingly, depending on mammal species a part of nucleosomes (e.g. 1% in mouse and 10-15% in human) remain unchanged. In this study we aimed to solve the meaning of nucleosome retention in sperm by analyzing nucleosome-binding sites in a genome-wide manner. METHODS Ten million sperm cells from a fertile donor were diluted in PBS and incubated with Lysolecithin. After DTT treatment, freed chromatin was digested with microccal nuclease. To separate nucleosomal (soluble) and protamine fractions (insoluble) freed chromatin was centrifuged. DNA isolated from nucleosomal fraction was separated on an agarose gel and the 146 bp mono-nucleosomal DNA was cutted out and purified. High throughput sequencing was performed using Genome Analyzer IIx (Illumina). Sequencing reads were aligned to the precompiled hg19 genome. All putative nucleosome-binding sites were analyzed considering coding and non-coding DNA (intergenic and intron), functional elements (promoter, 5’-UTR, 3’-UTR) and, moreover, repetitive DNA occurrence. RESULTS We found that in human sperm 5% of the genome, but not 10-15% as supposed before, is still bound to nucleosomes. Remarkably, sperm-nucleosomes remain mainly in centromere repeats, but also frequently in long and short interspersed nuclear elements (LINEs, SINEs). The majority of retained nucleosomes could be localized within non-coding intergenic and intron sequences. Analyzing gene regions we revealed that coding exons as well as transcriptional start and end sites are rather nucleosome-depleted. Gene promoters showed also a tendency of nucleosome-depletion, except some genes coding for cell-cell adhesion factors and calcium ion regulators. The main part of detected intragenic nucleosomes was scattered in gene bodies, interestingly often in genes coding for RNA- and protein-processing, and cell-cell adhesion factors. CONCLUSIONS Our results show that nucleosome-retention in sperm can be associated to certain genome regions and repetitive sequences. We suppose that the impact of sperm-nucleosomes is closely associated to post-fertilization centromere function but also to activity of retrotransposons. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e744 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Birgit Samans More articles by this author Yang Yang More articles by this author Stefan Krebs More articles by this author Helmut Blum More articles by this author Klaus Steger More articles by this author Wolfgang Weidner More articles by this author Temuujin Dansranjavin More articles by this author Undraga Schagdarsurengin More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Sperm Nuclear Basic Proteins of Tunicates and the Origin of Protamines

Sperm nuclear basic proteins (SNBPs) are the chromosomal proteins that are found associated with DNA in sperm nuclei at the end of spermiogenesis. These highly specialized proteins can be classified into three major types: histone type (H-type), protamine-like type (PL-type), and protamine type (P-type). A hypothesis from early studies on the characterization of SNBPs proposed a mechanism for the vertical evolution of these proteins that involved an H1 → PL → P transition. However, the processes and mechanisms involved in such a transition were not understood. In particular, it was not clear how a molecular transition from a lysine-rich protein precursor (H1 histone) to the arginine-rich protamines might have taken place. In deuterostomes, the presence of SNBPs of the H-type in echinoderms and of protamines in the higher phylogenetic groups of vertebrates had long been known. The initial work on the characterization of tunicate SNBPs attempted to define the types and range of SNBPs that characterize this phylogenetically intermediate group. It was found that tunicate SNBPs belong to the PL-type. In this work we discuss how the study of SNBPs in the tunicates has been key to providing support to the H1 → PL → P transition. Most significantly, it was in tunicates that a potential molecular mechanism to explain the lysine-to-arginine transition was first reported.

Role of Amino Acid Insertions on Intermolecular Forces Between Arginine Peptide Condensed DNA Helices: Implications for Protamine-DNA Packaging in Sperm

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. We have previously observed that the net attraction between salmon protamine condensed DNA helices was much smaller for DNA condensed by the equivalent homo-arginine peptide. We hypothesized that this is caused by the neutral amino acids present in protamines. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. The component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance have been determined by the osmotic stress technique coupled with x-ray scattering as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short-range repulsion; while only slightly decreasing the longer-ranged attraction. The decrease in net attraction between salmon protamine condensed helices compared with arginine homopeptides can be well explained by amino acid content alone. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both these observation have biological implications for protamine-DNA packaging in sperm heads.

Role of Amino Acid Insertions on Intermolecular Forces between Arginine Peptide Condensed DNA Helices: IMPLICATIONS FOR PROTAMINE-DNA PACKAGING IN SPERM

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.

Protamines: Structural Complexity, Evolution and Chromatin Patterning

Despite their relatively arginine-rich composition, protamines exhibit a high degree of structural variation. Indeed, the primary structure of these histone H1-related sperm nuclear basic proteins (SNBPs) is not random and is the depository of important phylogenetic information. This appears to be the result of their fast rate of evolution driven by positive selection. The way by which the protein variability participates in the transitions that lead to the final highly condensed chromatin organization of spermatozoa at the end of spermiogenesis is not clearly understood. In this paper we focus on the transient chromatin/nucleoplasm patterning that occurs in either a lamellar step or an inversion step during early and mid-spermiogenesis. This takes place in a small subset of protamines in internally fertilizing species of vertebrates, invertebrates and plants. It involves "complex" protamines that are processed, replaced, or undergo side chain modification (such as phosphorylation or disulfide bond formation) during the histone-to-protamine transition. Characteristic features of such patterning, as observed in TEM photomicrographs, include: constancy of the dominant pattern repeat distance λ(m) despite dynamic changes in developmental morphology, bicontinuity of chromatin and nucleoplasm, and chromatin orientation either perpendicular or parallel to the nuclear envelope. This supports the hypothesis that liquid - liquid phase separation by the mechanism of spinodal decomposition may be occurring during spermiogenesis in these species. Spinodal decomposition involves long wave fluctuations of the local concentration with a low energy barrier and thus differs from the mechanism of nucleation and growth that is known to occur during spermiogenesis in internally fertilizing mammals.

A Magnetofluorescent Nanoparticle for <I>Ex-Vivo</I> Cell Labeling by Covalently Linking the Drugs Protamine and Feraheme

We synthesized a nanoparticle (NP) for ex-vivo cell labeling and MRI tracking by covalently coupling the C-terminus of a rhodamine-labeled protamine (ProRho) to Feraheme (FH) in order to yield the nanoparticle denoted ProRho-FH. Since protamine can adsorb to certain charged surfaces, we confirmed a covalent interaction between ProRho and FH by heparin affinity chromatography. ProRho-FH lacks a net charge (zeta potential approximately 0) due to the combination of negative FH and positive ProRho charges. ProRho-FH was readily internalized by U87 cells and mouse mesenchymal stem cells as determined by FACS and MR relaxometry. Finally, some 4,000 stem cells were implanted in a mouse brain and imaged by MRI. Due to its lack of net surface charge, ProRho-FH relies on the internalizing properties of the surface guanidinium groups present in the arginine-rich protamine to induce NP uptake. ProRho-FH is a unique cell-labeling agent due to its synthesis using two approved drugs, magnetofluorescence, site-specific covalent attachment chemistry, and lack of surface charge.

Salmine and the homeotic integrity of early embryos of Norway spruce

Salmine, an arginine-rich protamine, is explored for its concentration-dependent potential to restructure the genome and remodel the homeotic development of Norway spruce embryos expressing monozygotic cleavage polyembryony (MCP). In controls and at low salmine, two protein fractions on SDS-PAGE gels were associated with cells responsible for generating the basal plan for early embryogenesis. With high salmine, embryonal initials no longer differentiated into embryonal tubes. Embryos having embryonal tubes no longer enucleated and differentiated into embryonal suspensors. Biomass and amino acid N declined. Nuclear and cytoplasmic organization was disrupted and nucleoli were highly vacuolated. The transcription of the two protein fractions, PCNA (cyclin) activity and MCP were blocked. Cellular proteins were turned over by proteasomal ubiquitination and others released into the culture medium. Biomass loss and gluconeogenesis of amino acids led to the accumulation of free arginine N. No evidence was obtained with salmine for the remodeling of cells into gametes.

The Sperm Proteins from Amphioxus Mirror Its Basal Position among Chordates and Redefine the Origin of Vertebrate Protamines

The sperm nuclear basic proteins (SNBPs) that participate in chromatin condensation in spermatozoa belong to 3 groups: histone (H), protamine-like (PL), and protamine (P) type. They share a common origin with histone H1 resulting from the segregation of PL components, corresponding to different regions of an H1 precursor molecule (N-terminal, winged-helix, C-terminal domains), becoming independent and following a subsequent process of parallel vertical evolution (H <--> PL <--> P). In the present work, we describe the sequence and primary structure of the main SNBP component in the sperm of the cephalochordate Branchiostoma floridae (amphioxus), revealing that it represents the deuterostome counterpart of the PL-III SNBP component from molluscs corresponding to the H1 N-terminal region. Until now, this has been a missing piece needed to complete the evolutionary history of SNBPs in metazoan genomes. The discovery of this PL lineage in deuterostomes definitively validates the parallel vertical evolution of SNBPs across metazoans, giving further support to the "basal" position of amphioxus among chordates, with respect to tunicates. Sequence analyses suggest that later on in evolution, the appearance of positively selected arginine-rich protamines, derived from the H1 C-terminal region, led to the extinction of this PL lineage in the genomes of early protostomes and deuterostomes. Given that tunicates are now viewed as a sister group of vertebrates, the lysine to arginine transition responsible for the origin of vertebrate protamines must be set a step back from tunicates.

Histone H1 and the origin of protamines.

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicate Styela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate, Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.

Electrochemical Assay of Plasminogen Activators in Plasma Using Polyion-Sensitive Membrane Electrode Detection

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these “clot-busting” drugs.

Sperm nuclear maturity in spinal cord-injured men: Evaluation by acidic aniline blue stain

For many of the spinal cord-injured (SCI) men who are able to produce an ejaculate, infertility because of poor semen quality is a concern. Impaired spermatogenesis has been attributed as a possible reason for the poor semen quality. If so, events that occur during spermatogenesis may be used as a marker to evaluate the extent of spermatogenic alteration. During spermatogenesis, when the sperm nuclear condensation occurs, lysine-rich somatic histone are replaced by arginine-rich protamines in the DNA. Acidic aniline blue preferentially stains the immature sperm nucleus blue by binding to the lysine. Hence, each sperm can be individually evaluated for nuclear maturity. To test this concept, the nuclear maturity of sperm from 12 SCI men obtained by vibratory stimulation was compared with sperm samples obtained by self-masturbation from 104 non-SCI men. Sperm smears stained with acidic aniline blue were evaluated for nuclear maturity. The percent of unstained spermatozoa for non-SCI men (mean ± SEM; 83.4 ± 1.1%) was not statistically different from that of the SCI men (79.7 ± 4.8%). However, the sperm motility (70.5 ± 1.2%) and the percentage of normal sperm morphology (50.8 ± 0.7%) of non-SCI men were significantly ( P < .01) different from those of the SCI men (36.5 ± 6.8% and 44.0 ± 2.4%). It seems that the poor semen quality observed in SCI men is probably not caused by inadequate nuclear maturity of the spermatozoa.

Complement C5a Receptor Antagonism by Protamine and Poly-L-Arg on Human Leukocytes

It is shown that protamine selectively and dose-dependently inhibits complement C5a-induced leukocyte responses such as histamine release from basophils, chemiluminescence and beta-glucuronidase release from neutrophils. Protamine produces parallel rightward displacements of the C5a dose-response curves. The inhibitory capacity of the polypeptide is reversible and disappears following repeated washing of exposed cells. In neutrophils poly-L-Arg similarly and specifically antagonizes C5a-induced chemiluminescence and enzyme release. This polymer alone, however, degranulates basophils and neutrophils, leading to histamine and enzyme release, respectively. It is concluded that on human neutrophils the arginine-rich polycations protamine and poly-L-Arg exhibit a competitive C5a receptor antagonism. In addition, protamine inhibits the C5a receptors on basophils. It is hypothesized that molecular conformations of the arginine-rich polycations might bind reversibly to, and block negatively charged groups at the C5a-receptor sites.

Extraction purification and characterization of the sperm protamines of the dog-fish Scylliorhinus caniculus

Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with othe sperm-specific protamines previously described.

Release of low density lipoprotein receptors from human fibroblasts or Hela cells by tryptic digestion

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with 6-[ 3H]-glucosamine·HCl and [ 3H ]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.

Acrolein-Schiff, a new staining method for proteins.

A cytochemical method based on the reaction of acrolein with tissue compounds followed by staining with the Schiff reagent is described. The reaction is completely blocked by acetylation. Filter paper spot tests of carbohydrates, nucleic acids and proteins revealed a high specificity for proteins. All proteins except the argininerich protamines were positive in the concentrations tested. Carbohydrates, as well as ribonucleic acid, were negative; deoxyribonucleic acid reacted weakly, probably as a result of a contamination. Tests with compounds of low molecular weight revealed that amino acids were positive, nucleic acid derivatives negative. Arguments are given that the double bond of acrolein reacts with SH, with aliphatic NH2 and NH groups, and with the imidazole group. The use of this method as a cytochemical stain in combination with Feulgen, thionine-SO2 staining is indicated, especially for chromosome studies. The method also seems applicable to paper and agar electrophoresis.