Interaction of fibrinogen and fibrin with protamine sulfate

Fibrinogen and fibrin sedimentation by different protamine sulphate preparations have been studied. Ionic strength and protamine sulphate concentration are found to influence the sedimentation reaction (paracoagulation). High sedimentation activity is inherent in protamine sulphate preparations with the lower electrophoretic mobility, that is with the higher molecular weight. The protamine sulphate reaction with fibrinogen and fibrin is of electrostatic character as the long polycationic chain of protamine is coupled with the negatively charged loci either of fibrin or of fibrinogen molecules, thus evoking aggregation. In this case the fibrin molecules being brought together favour the specific mutual binding due to the active sites of polymerization, specific fibres or gel being formed.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.     

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

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.     

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.     

Purification and properties of a distinct protamine kinase from the cytosol of bovine kidney cortex

A protamine kinase has been purified to apparent homogeneity from extracts of the cytosol of bovine kidney cortex. This protamine kinase exhibited an apparent Mr = 43,000 as estimated by gel permeation chromatography on Sephacryl S-200 and an apparent Mr = 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protamine kinase exhibited about 5% activity with casein, 8% with histone H2B, and less than 0.1% with histone H1, histone H4, glycogen synthase a from rabbit skeletal muscle, ovalbumin, bovine serum albumin, and phosvitin. The activity of the highly purified protamine kinase was unaffected by cyclic AMP (up to 0.1 mM), cyclic GMP (up to 0.1 mM), the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase (up to 100 micrograms/ml), heparin (up to 100 micrograms/ml), EGTA (up to 1 mM), Ca2+ (up to 1 mM), calmodulin (up to 0.5 microM) in the absence or presence of Ca2+ (0.05 mM), and phosphatidylserine (up to 40 micrograms/ml) and/or diolein (up to 1 microgram/ml) in the absence or presence of Ca2+ (up to 0.5 mM). Experiments in which extracts of kidney cytosol were incubated with [gamma-32P]ATP and MgCl2 revealed that the phosphorylation of numerous polypeptides was markedly increased in the presence of the purified protamine kinase. The results indicate that this protamine kinase of kidney cytosol is a novel protein kinase.     

Amino acid composition, heterogeneity and antiheparin activity of protamine sulfate from the milt roe of the sturgeon Acipenser sturio

The homogeneous preparation of protamine sulphate is obtained chromatographically and electrophoretically from milt roe of the sturgeon. Its amino acid composition and properties are studied. The methods to blockade the functional groups of protamine sulphate amino acids is used to investigate the possible mechanism of heparin inactivation.     

Heterogeneity of biologically active deadenylated protamine mRNA components isolated from rainbow trout testes.

Poly(A)+ protamine mRNA's were isolated from rainbow trout testes and deadenylated by treatment with calf thymus RNase H. Four subcomponents of deadenylated PmRNA (PmRNA1-4) were purified by electrophoresis on a 6% polyacrylamide gel in 8 M urea. Translation of each PmRNA subcomponent in the wheat germ S-30 cell-free system showed that all subcomponents are biologically active but each codes for two or more protamine polypeptides suggesting molecular heterogeneity. However, the deadenylated mRNA's can be categorized into two groups based on the spectrum of protamines whose synthesis they stimulate.     

Inhibition of pepsin by polyions and c.d. studies

The effects of poly(vinyl sulphate) (PVS), poly(vinyl pyrrolidone) (PVP) and protamine sulphate with the enzyme pepsin (EC 3.4.23.1) have been investigated. PVS, PVP and protamine acted as inhibitors of the enzyme pepsin at low concentrations, but at high concentrations of the polyions (with the exception of PVS) the inhibition was less pronounced. The catalytic effectiveness of several polyions has been shown experimentally; when used at pH 2.1 with haemoglobin and at pH 5.0 with azocasein thus acted as a weak proteolytic catalyst. The order of effectiveness was: polybrene greater than poly(L-lysine) (PLL) greater than spermine greater than spermidine greater than protamine greater than and PVP relative to pepsin. The hydrolysis of the substrates by the enzyme decreases in the presence of high concentrations of monovalent and/or divalent salts. The purity of the enzyme was assessed by determination of the molecular weight using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), phosphorus and nitrogen content. The circular dichroism (c.d.) spectrum of the enzyme in the wavelength range 240-310 nm, where the polycations did not have a c.d. spectrum has also been studied. Each of the polyions had a definite effect on the c.d. spectrum, showing strong binding to the enzyme. The neutral polymer PVP was also found to modify the c.d. spectrum, showing strong binding to the enzyme. The strengths of the interactions as indicated by the magnitudes of the changes in the c.d. spectrum of pepsin at 270 nm were: polybrene greater than protamine greater than PLL greater than PVP greater than spermidine greater than spermine.     

Nucleotide sequence of a bovine protamine cDNA

The nucleotide sequence of a 441-base cDNA encoding the bovine protamine has been determined. This insert, isolated from a bovine spermatid-specific cDNA library, encodes a polypeptide of 50 amino acids of which 26 are arginine, 7 are cysteine, and 2 are tyrosine. The insert contains the complete 3'-noncoding region of 150 bases and most of the 5'-noncoding region. The predicted amino-acid sequence of bovine protamine is about 96% homologous to ram protamine, 76% to boar protamine, 64% to mouse protamine 1 and 52% to human protamine 1 and contains the central, highly basic domain of four arginine clusters found in the trout protamines. Our results show that bovine protamine is 50 amino-acid residues in length and not 47 residues as previously published (Coelingh, J.P. et al. (1972) Biochim. Biophys. Acta 285, 1-14).     

Amino acid sequence of the unique protamine from yellow perch.

By the criteria of gel electrophoresis, ion-exchange chromatography, and reverse-phase HPLC, yellow perch protamine behaves as a single component. This observation was confirmed by automated Edman degradation which gave a single unambiguous amino acid sequence PRRRRHAARPVRRRRRTRRSSRVHRRRRAVRRRR. Yellow perch protamine has 34 amino acids, including 21 arginines. It has two histidines, neither of which interrupts an arginine tract. It is unusual among fish protamines in not having a serine or threonine N-terminal to the second arginine tract, and is unique in not being a mixture of components.     

Electrostatic interactions in the early events of VSV infection

The inportance of electrostatic interactions in the early phases of vesicular stomatitis virus (VSV) infection has been investigated in susceptible cells of different origin, human (HeLa) and avian (CER), by using some polyanions (heparin, polygalacturonic acid and mucin) and polycations (polymyxin B sulphate, poly-L-lysine, protamine, histone and polybrene). In HeLa cells, the attachment of VSV was enhanced by polymers having a positive charge and inhibited by those having a negative charge. In CER cells, all the polyanions tested reduced virus infection. Among the polycations, histone, polymyxin B sulphate and poly-L-lysine enhanced virus plaque forle protamine and polybrene reduced virus attachment. The effect of polyions on VSV particles and on cell membrane receptors has also been investigated. The analysis of the results obtained suggest that, although electrostatic interactions play an essential role in the binding of VSV to the cell membrane, more specific structural features appear to be required for viral attachment to occur.     

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.     

Divergence of protamine gene sequences in fish

Complementary DNA to trout protamine mRNA was hybridized to excess genomic DNA from trout, salmon and yellow perch. Although there was extensive hybridization of the cDNA to trout DNA, no cross-reaction with yellow perch DNA was observed and the hybridization to salmon DNA was noticeably less than in the homologous reaction. To confirm these results, yellow perch protamine mRNA was purified and compared directly to trout protamine mRNA. Yellow perch protamine mRNA was shorter than trout protamine mRNA, when measured by agarose gel electrophoresis in the presence of methyl mercury hydroxide. The two mRNAs did not cross-react in cDNA/RNA hybridizations, although the homologous reactions went to 90% of completion. This lack of sequence homology was confirmed when the oligopyrimidine tracts from the cDNAs were compared. No sequences longer than tetranucleotides were common to both species. Trout protamine cDNA contained oligopyrimidines of composition C₇T₄, C₄T₂, C₃T₂, C₂T₄, C₂T₃, C₁T₅ and C₁T whereas yellow perch protamine cDNA contained C₆T₃ and C₄T₃.     

Purification and characterization of a novel protamine kinase in HL60 cells

A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.     

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.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Human sperm protamines. Amino-acid sequences of two forms of protamine P2

Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium X HCl containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys-C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gas-phase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protamine P1. The amino acid sequence of P2a is: (sequence; see text)