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PubMed1984

Protamine: a powerful in vivo inhibitor of bone resorption.

Protamine is shown to be a powerful disrupter of calcium homeostasis, acutely inducing a severe hypocalcemia in both rabbits and rats. The magnitude of its effect correlates with bone turnover. Protamine does not significantly alter the renal excretion of calcium, and is effective whether or not there is calcium present in the gut. Protamine causes a significant fall in the specific activity of 45Ca in the blood in animals whose bone has been prelabeled with 45Ca. These data suggest that protamine induces hypocalcemia by blocking calcium efflux from bone. Further work seems indicated to define the biochemical mechanism of this action.

Animals↗
PubMed1985

Thrombocytopenia accompanying a reaction to protamine sulfate.

Catastrophic pulmonary vasoconstriction, an idiosyncratic reaction to protamine, occurred following cardiopulmonary bypass in a patient with mitral regurgitation. One minute following intravenous injection of 10 mg of protamine, systemic blood pressure decreased precipitously, accompanied by elevated pulmonary arterial pressure, decreased left atrial pressure, and a dilated and poorly contracting right ventricle. These changes spontaneously reversed over five minutes. Rechallenge with protamine was uneventful. Platelet count decreased from 76,000/mm3 to 12,000/mm3. Transient thrombocytopenia occurs predictably in dogs given protamine rapidly. Suggested mechanisms of idiosyncratic pulmonary vasoconstriction in patients should consider the role of the associated thrombocytopenia.

Aged↗
PubMed1986

Immobilization of uricase on protamine bound to glass beads and its application to determination of uric acid.

Uricase was found to be stabilized by protamine from salmon testis. Protamine was then bound to controlled-pore glass beads aminohexyl CPG 500 using glutaraldehyde. Microbial uricase was readily immobilized on the protamine bound to glass beads. The immobilized uricase proved to be stable even at 70 degrees C, whereas free uricase was inactivated at 45 degrees C and showed activity over a broader pH range than free uricase. Automated analysis of uric acid was facilitated using the immobilized uricase. The standard curve for uric acid was linear in the range of 2 to 10 micrograms/sample and passed through the origin. This automated procedure was also applicable to the determination of uric acid in human serum. Protamine bound to glass beads is expected to be useful for the simple immobilization and stabilization of enzymes.

Animals↗
PubMed1987

Phosphorylation of protamines by protein kinase C: involvement of sites which are phosphorylated in vivo and are not affected by cAMP-dependent protein kinase.

Most fish protamines contain two phosphorylatable sites both of which incorporate phosphate in vivo. Here we show that in two protamines (salmine A1 and clupeine Y1) the site more distant from the N-terminus (residues 20-21) is unaffected by cAMP-dependent protein kinase while it represents the main target for protein kinase C. Such a phosphorylation is typically independent of Ca2+ and phospholipids: responsiveness to these effectors however is conferred by previous fragmentation of protamine with thermolysin. These results suggest that Ca2+, phospholipid-independent phosphorylation of protamine by protein kinase C might have physiological relevance and shed light on the structural basis for the specificity of such an unique process.

Amino Acid Sequence↗
PubMed1983

Effect of protamine sulfate on antithrombin III activity.

When protamine sulfate was added to heparinized plasma in vitro for neutralization of heparin, the activities on both thrombin and Xa known as heparin cofactor in antithrombin action were completely abolished. However, progressive activities on thrombin and Xa both recovered within 30 minutes after protamine sulfate addition. When equivalent heparin was again added, heparin cofactor activity was immediately restored. Based on the fact that protamine sulfate did not show any direct action on the antithrombin III molecule, the presence of AT III with progressive activity was considered to play an important role in the rebound phenomenon of heparin after heparin neutralization with protamine sulfate.

Antithrombin III↗
PubMed1988

Haploid expression of the rooster protamine mRNA in the postmeiotic stages of spermatogenesis.

cDNA clones were prepared from poly(A)+ mRNA isolated from a population enriched in postmeiotic rooster testes spermatogenic cells. A series of clones was sequenced at random and two partial sequences corresponding to the C-terminal coding and 3' untranslated region of the chicken protamine mRNA were obtained. The deduced amino acid sequence of this C-terminal coding region corresponds to the sequence previously described at the protein level for the chicken protamine, galline [Nakano, M., Tobita, T., and Ando, T. (1976), Int. J. Peptide Prot. Res. 8, 565-578]. To study the expression of this protamine gene, RNA was prepared from chicken testes at different stages of development, electrophoresed in formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with a rooster protamine cDNA probe. Two populations of mRNA of sizes ranging between 420 and 465 bases are expressed in postmeiotic rooster testis cells. To determine if there was a differential expression of the two populations of mRNA in the final postmeiotic haploid stages of spermatogenesis, RNA was purified from adult rooster cells separated at unit gravity according to their differences in size by the Staput technique. The RNA was similarly analyzed by Northern blots. The results indicate that round spermatids are enriched in the 465-nucleotide mRNA species, whereas in the final stage of elongated spermatids the 420-nucleotide species is the only one present, suggesting either post-transcriptional processing, the presence of two different sets of genes that are differentially expressed, or a single set of genes with differential promoter usage.

Amino Acid Sequence↗
PubMed1989

Comparison of partial amino acid sequences of two protamine 2 variants from stallion sperm. Structural evidence that the variants are products of different genes.

Protamine 1 and two protamine 2 variants were isolated from stallion sperm and separated by acetic acid-urea gel electrophoresis. After electroblotting onto polyvinyldifluoride filters, their amino-terminal amino acid sequences were determined by pulse-liquid peptide sequencing. The sequences of the two protamine 2 variants are homologous but slightly different in length and amino acid composition and indicate for the first time the existence of two different genes for this protamine species.

Amino Acid Sequence↗
PubMed1991

Partial in vivo neutralisation of plasma anticoagulant effects of Lomoparan (Org 10172) by protamine chloride.

In a cross-over study increasing doses of protamine hydrochloride (20-100 mg) or placebo were administered to six groups of four healthy male volunteers each, following a single intravenous dose of 3200 anti-Xa units of Org 10172. No neutralising effects were observed on the Org 10172 induced changes in the bleeding time, prothrombin time and thrombin time. A small and statistically not significant temporary decrease in anti-Xa activity was observed after doses of 80 and 100 mg protamine chloride. The anti-thrombin activity was dose-dependently and partly irreversibly neutralised by protamine chloride to a maximum of approximately 60%. This neutralisation correlated with the observed shorter prolongation of the thrombin time. The thrombin-generation inhibition activity was for approximately 35% neutralised by protamine chloride doses of 60-100 mg.

Adolescent↗
PubMed1992

Effects of aprotinin on heparin activities and heparin neutralization with protamine.

In order to investigate the new situation in which aprotinin is proposed as a novel approach to reducing post operative bleeding, specially in cardiopulmonary bypass (CPB) surgery during which heparin and protamine are commonly used, preliminary in vitro and in vivo studies have been performed. Aprotinin increases the anticoagulant heparin effects in vitro, and the hemorrhage time in vivo. But in addition to protamine, there are no statistically significant differences with heparin-protamine situation, indicating aprotinin does not disturb the neutralizing activities of protamine on heparin.

Animals↗
PubMed1992

Ex vivo activity of heparin is not predictive of blood loss after neutralization by protamine.

To study whether the ex vivo activity of heparin and Fraxiparin correlates to and predicts the extent of blood loss induced by the heparins (pre- and post neutralization by protamine), a rat tail transection model and a rabbit ear bleeding model were used. In the rat model heparin (2 mg/kg i.v.) significantly prolonged the bleeding time, while this dose of Fraxiparin had no effect. In the rabbit ear blood loss model, heparin (2 mg/kg i.v.) produced significant increases in blood loss while Fraxiparin (2 mg/kg i.v.) produced approximately 30% of the blood loss induced by heparin. Equigravimetric protamine reduced the heparin-induced blood loss by approximately 50%, however, significant blood loss, thrombin time and Heptest activity remained. Heparin and Fraxiparin (3 mg/kg s.c.) did not cause any increased bleeding. While, all activities of heparin were completely neutralized by protamine, the Heptest activity of Fraxiparin was resistant to neutralization. The ex vivo activity of heparins after neutralization by protamine does not correlate to the extent of blood loss which suggests it may not be necessary to neutralize all ex vivo activities of the heparins to baseline values to be assured that blood loss is reversed.

Animals↗
PubMed1988

Detection of the major epitopes of human protamine P1 recognized by rabbit and mouse antibodies.

The characterization of the major antigenic determinants present in human protamine P1 has been carried out by the use of specific rabbit polyclonal and mouse monoclonal antisera raised against protamine P1. This basic protein, the full amino acid sequence of which has been determined here, has been cleaved by cyanogen bromide and/or by pepsin to generate a discrete number of peptides. These have been purified, characterized by partial amino acid sequencing and used for the determination of their antigenic reactivities with antisera to native protamine P1. Both rabbit polyclonal and mouse monoclonal antibodies were able to recognize the NH2-terminal CNBr peptide encompassing residues 1-36 to the same extent as the intact protamine. A minor epitope present on the COOH-terminal peptide 37-50 could be detected only with the polyclonal rabbit antisera. Attempts to further cleave the P1 molecule in order to isolate peptides shorter than fragments 1-36 whilst retaining full antigenic reactivities, were unsuccessful. This suggests that the epitopes in P1 are conformation-dependent and located for the most part on the amino-terminal half of the molecule, which comprises the characteristic central arginine cluster. The implication of these findings for the studies of the specificities of autoantibodies in sera from infertile and vasectomized individuals is discussed.

Amino Acid Sequence↗
PubMed1983

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 other sperm-specific protamines previously described.

Amino Acids↗
PubMed1986

A corrected primary sequence for bull protamine.

We have redetermined the primary sequence for bull protamine using HPLC peptide mapping and automated amino-acid sequencing techniques and report, on the basis of these findings, that the previously published amino-acid sequence for this protein is incorrect. The correct protamine sequence is 50 amino acids in length and differs from the original published sequence by the tripeptide -Cys-39-Arg-40-Arg-41-. Analyses of protamine tryptic peptides derived from nine diverse breeds of Bos tarus and Bos indicus indicate that this sequence is present in the protamine of each breed and that it does not represent a variant or mutation.

Amino Acid Sequence↗
PubMed1993

Structure and function of protamines: an 1H nuclear magnetic resonance investigation of the interaction of clupeines with mononucleotides.

Protamines form a class of low-molecular-weight proteins that protect the chromosomal DNA in the spermatic cells of eukaryotic organisms. Protamines are located in the small and/or large groove of DNA where they complex the DNA nucleotides. Very little is known up to date on the role and specificity of binding of the various protamine fractions belonging to a single eukaryotic species. In the present paper, a detailed investigation on the complexation properties of the protamine fractions (clupeines) extracted from herrings has been carried out by means of proton nuclear magnetic resonance and ultraviolet absorbtion data. In particular, the binding properties of the clupeine fractions with purinic (5'dAMP) and pyrimidinic (5'dCMP) mononucleotides have been measured and analysed at different clupeine concentrations. The results indicate that, contrary to previous preliminary hypothesis, the three clupeine fractions exhibit quite comparable binding properties toward mononucleotides. In addition it has been found that nucleotides can induce a conformational transition of the disorder-order type in the clupeine molecules and this property is concentration and temperature dependent. It is concluded that, as far as specificity is concerned, the clupeine fractions seem to possess the same behaviour toward mononucleotides.

Amino Acid Sequence↗
PubMed1986

A comparison of the strength of binding of antithrombin III, protamine and poly(L-lysine) to heparin samples of different anticoagulant activities.

The limiting concentrations, i.e., those concentrations of sodium chloride required to completely disrupt the complexes of heparin with antithrombin III, protamine and poly(L-lysine), were determined using fluorescence techniques, in order to compare the binding strengths of these complexes. From the limiting salt concentration values, poly(L-lysine) always exhibited stronger binding to heparin of a particular anticoagulant potency (degree of sulphation) than did protamine. The binding strengths of both complexes decreased as the degree of sulphation of the heparin participating in the complex was reduced. In contrast, the limiting salt concentration values for complexes formed between antithrombin III and heparin did not change with either the degree of sulphation or the biological potency of the heparin samples. A low-potency heparin simply contained a smaller number of molecules which possessed the intact antithrombin III binding site (thus being fully 'anticoagulant active') than a high-potency sample. Low-affinity heparin did not contain these binding sites and thus showed a low affinity for antithrombin III. High-potency heparin, being highly sulphated, possessed a higher affinity for protamine and poly(L-lysine) than for antithrombin III. However, after partial N-desulphation of heparin, the subsequent heparin-protamine complex was more weakly bound than a significant proportion of the corresponding heparin-antithrombin III complexes. These in vitro findings may have particular relevance in relation to the clinical condition termed 'heparin rebound'.

Anticoagulants↗
PubMed2006

Identification of novel polymorphisms in the nuclear protein genes and their relationship with human sperm protamine deficiency and severe male infertility.

OBJECTIVE: To screen populations of fertile men, severely infertile men, and infertility patients with aberrations in the protamine-1-to-protamine-2 ratio (P1/P2) for clinically relevant alterations in the P1, P2, transition protein 1 (TP1), and transition protein 2 (TP2) genes. DESIGN: Prospective case-control study. SETTING: University-based infertility clinic. PATIENT(S): Ninety-six fertile men, 96 severely infertile men, and 96 infertility patients with aberrations in the sperm P1/P2 ratio. INTERVENTION(S): Sperm nuclear protein extraction, gel electrophoresis, and densitometry to evaluate the P1/P2 ratios of infertility patients. DNA extraction, polymerase chain reaction (PCR), and gene sequencing to screen the nuclear protein genes. MAIN OUTCOME MEASURE(S): Sperm P1/P2 ratios and single nucleotide polymorphisms (SNPs) in the sperm nuclear protein genes (P1, P2, TP1, and TP2). RESULT(S): Fifteen SNPs were identified in the nuclear protein genes, 12 of which were previously unreported. Five variants were identified that resulted in amino acid changes (one in P1, one in TP1, and three in TP2). The frequency of the 15 SNPs was similar in protamine-deficient patients, severely infertile patients, and fertile controls. CONCLUSION(S): A number of SNPs are present in the testis-specific nuclear protein genes. However, the gene variants do not appear to underlie protamine deficiency and severe male infertility.

Adult↗
PubMed2006

The probable structure of the protamine-DNA complex.

A detailed molecular structure is proposed for the human protamine-DNA complex, which has hitherto been largely a mystery. The structure was created with virtual modeling software (AmiraMol), employing logical deduction as the primary investigative tool. A beta-sheet structure for the protein component is essentially mandated, as the alternatives can be decisively excluded. A dimeric structure too is essentially mandated, since the cysteine residues of protamines P1 and P2 are invariably aligned in all species having both chains. The cross-sectional and axial spacings of arginine guanidinium groups in this protein structure can be perfectly aligned with those of phosphate groups in DNA according to the DNA structure proposed by Wu. This is a non-helical structure, whose possible occurrence in certain plasmids has been suggested by experimental observations. The unit cell of this protamine-DNA complex is essentially devoid of steric hindrances, and heavily favored by a multitude of ionic and hydrogen bonds. The packing of adjacent "unit cells" of the protamine-DNA structure is based on a complex array of salt bridges, the mere existence of which is so fortuitous that it is virtually inconceivable that it comes about through a mere modeling "coincidence". The possible significance of the structure beyond the sperm cell is discussed.

Amino Acid Sequence↗
PubMed2003

Dextran sulfate included in factor Xa assay reagent overestimates heparin activity in patients after heparin reversal by protamine.

A lack of correlation between activated partial thromboplastin time (aPTT), thrombin time (TT) and anti-factor Xa (AXa) activity was observed in patients after cardiac surgery with cardiopulmonary bypass (CBP). Indeed, AXa activity measured by the chromogenic assay, Coamatic Heparin, was higher than expected with regard to results obtained in coagulation assays. To account for this discrepancy, another AXa chromogenic assay was tested. First, AXa activity was measured with two chromogenic assays (Coamatic Heparin and Rotachrom Heparin) in plasma samples of 25 patients undergoing cardiac surgery at two time points after heparin reversal by protamine. AXa activity was significantly higher when measured with Coamatic Heparin than with Rotachrom Heparin in samples collected just after protamine infusion (p<0.01). Next, since Coamatic( Heparin contains dextran sulfate (DXS) to reduce the influence of heparin antagonists such as platelet factor 4 (PF4), whereas Rotachrom Heparin does not, we hypothesized that the dextran sulfate contained in the reagent might explain this discrepancy. We therefore performed in vitro studies consisting in neutralizing unfractionated heparin (UFH) with protamine and measuring AXa activity with the two chromogenic assays. An AXa activity was still measurable with Coamatic Heparin after neutralization, thus strongly suggesting that dextran sulfate dissociates protamine/heparin complexes. We conclude that Coamatic Heparin assays should be avoided when measuring AXa activity in plasma samples immediately after protamine infusion, as inaccurate results may lead to inadequate management of heparin reversal.

Anticoagulants↗