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PubMed2000

Early mobilization after protamine reversal of heparin following implantation of phosphorylcholine-coated stents in totally occluded coronary arteries.

Immediate removal of the femoral artery sheath after coronary angioplasty may allow rapid mobilization and reduces the number of in-hospital days. We studied the early and 1-month clinical and angiographic follow-up of patients having heparin reversed with protamine after implantation of phosphorylcholine-coated metal (Divysio) stents, followed by removal of the femoral artery sheath. Fifty patients (37 men, mean age 59 +/- 10 years) with stable angina pectoris and a single totally occluded artery (1 unprotected left main stem, 15 left anterior descending, 11 left circumflex, 23 right) underwent coronary angioplasty. Antithrombotic medication was salicylic acid 75 to 160 mg before, heparin bolus 7,500 IU during, and protamine sulfate 25 mg and oral ticlopidine 250 mg after the procedure. Angiography was performed after 30 minutes and at 1 month. The mean number of stents was 1.4 +/- 0.6/lesion, with a mean final diameter of 2.69 +/- 0.40 mm. One stent thrombus was detected after 30 minutes and was treated with balloon dilatation. One patient underwent emergency bypass surgery for non-stent-related problems. Forty-six patients were mobile after 5 hours, and 2 after >5 hours. At 1 month there had been no major coronary end points, rehospitalizations, groin bleeding, or more thrombi. One episode of transient pulmonary edema occurred after protamine injection. Thirty-eight patients (79%) had no angina at 1 month, maximal bicycle exercise capacity increased from 128 +/- 42 to 160 +/- 45 W (p <0.05), and left ventricular ejection fraction increased from 63% to 68% (p <0.05). Thus, reversal of heparin with protamine sulfate after implantation of a phosphorylcholine-coated stent enables early mobilization. This approach seems safe in patients with 1 -vessel total occlusions and angioplasty could be performed as an outpatient procedure.

Angioplasty, Balloon, Coronary↗
PubMed1999

Characterization of aneuploidy rates, protamine levels, ultrastructure, and functional ability of round-headed sperm from two siblings and implications for intracytoplasmic sperm injection.

OBJECTIVE: To characterize and contrast protamine levels, chromatin decondensation, chromosome aneuploidy rates, and functional ability of round-headed sperm from two brothers with round-headed sperm syndrome. DESIGN: Analysis of semen samples from siblings with round-headed sperm syndrome and comparison with semen of fertile donors. SETTING: University school of medicine and laboratories. PATIENT(S): Two infertile siblings. MAIN OUTCOME MEASURE(S): Sperm aneuploidy rates of chromosomes X, Y, 13, 18, and 21, protamine 1 and 2 (P1-P2) ratios, Western blot evaluation of protamines, and chromatin decondensation rates. Additional measures include standard semen quality parameters, electron microscopy ultrastructure evaluation, analysis of acrosome morphology, and sperm penetration rates. RESULT(S): Aneuploidy rates were significantly increased in sibling no. 1 but not in sibling no. 2. The levels of P1 and P2 were decreased in sibling no. 1, and an unusual level of protamine precursors was present. Ultrastructural differences also were observed between the siblings. CONCLUSION(S): These data indicate profound differences in sperm from two siblings with complete round-headed sperm syndrome. Multigenic defects and/or variable expression of the syndrome may be responsible for the syndrome and necessitate individual screening of affected individuals because the pattern of expression appears highly variable.

Acrosome↗
PubMed1997

The effect of protamine sulphate on plasma tissue factor pathway inhibitor released by intravenous and subcutaneous unfractionated and low molecular weight heparin in man.

Heparin, a negatively charged sulphated glycosaminoglycan, is clinically the most important antithrombotic drug. Heparin augments the inhibitory activity of antithrombin (AT) towards thrombin, factor Xa (FXa) and other activated clotting enzymes. Tissue factor pathway inhibitor (TFPI) is an endogenous heparin releasable three domain Kunitz-type coagulation inhibitor which inhibits the crucial tissue factor-factor VIIa (TF-FVIIa) dependent coagulation pathway in the presence of FXa. The importance of the TF-FVIIa pathway and TFPI has recently been reviewed (1). TFPI is located to different vascular pools, the largest being the vascular endothelium from where TFPI can be released dose-dependently to the blood by heparins (2). TFPI is speculated to contribute to the anticoagulant properties of heparins, but to which degree is not yet fully understood. In recent years low molecular weight heparins (LMWH) have proven to be effective and safe both for prophylactic (3) and therapeutic treatment (4) of deep vein thrombosis (DVT). Protamine is the least toxic and clinically most commonly used antidote to heparin. However, in vitro and in vivo LMW heparinized blood is not fully neutralized by protamine, as substantial anti-Xa activity remains following neutralization (5). This post-protamine effect has been shown to be partly TFPI dependent when measured in a dilute TF-dependent assay (6,7). We undertook this in vivo study on healthy volunteers in order to investigate whether TFPI released by UH or LMWH (intravenous (iv) or subcutaneous (sc)) remains in the circulation following neutralization of the heparin activity with protamine sulphate (PS). We measured TFPI by three different methods-chromogenic activity, anticlotting activity and a new antigen assay specific for full-length and three-domain TFPI.

Age Factors↗
PubMed1999

Protamine allergy as a complication of insulin hypersensitivity: A case report.

BACKGROUND: Although most patients receiving insulin produce insulin-specific IgE, significant allergic symptoms develop in very few of them. Patients receiving neutral protamine Hagedorn (NPH) insulin are at increased risk for the development of protamine hypersensitivity. The case of a 19-year-old woman with insulin-dependent diabetes and regular and NPH insulin hypersensitivity is presented. OBJECTIVE: The purpose of this study was to determine whether desensitization to NPH insulin, as well as standard insulin desensitization, could control allergic symptoms in a patient allergic to both NPH and regular insulin. METHODS: The patient required insulin desensitization for severe urticaria, angioedema, and occasional wheezing resulting from her insulin dose. She underwent a standard protocol for insulin desensitization twice in a 2-month period, with persistence in her symptoms. She was found to have high protamine-specific, as well as insulin-specific, IgE levels, and because of her poor response to regular insulin desensitization, she was desensitized to both regular and NPH insulin. RESULTS: Dual desensitization resulted in marked improvement in her symptoms. The patient had recurrence of urticaria and angioedema a year and a half later, at which point the NPH was stopped and she was desensitized to regular insulin. She continued to receive regular insulin 4 times per day over the following 3 years with only occasional hives. CONCLUSION: Patients with insulin allergy may not have complete resolution of their symptoms after standard desensitization, particularly those patients with concomitant protamine allergy. These patients may require protamine/NPH desensitization, an alternative insulin preparation, or both.

Adult↗
PubMed1997

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

Amino Acid Sequence↗
PubMedDate not supplied

Allergy to protamine sulfate.

Adverse responses to protamine sulfate have been identified for many years. The antigen-antibody response to protamine sulfate results in a type I anaphylactic reaction. Manifestations of allergic reactions include hypotension, bronchospasm, and skin and mucous membrane reactions. The severity of the adverse responses may vary from mild to causing death. Several potential risk factors for adverse reactions to protamine have been identified, including insulin-dependent diabetes mellitus, vasectomy, allergy to fish, prior exposure to protamine sulfate, and the rate of infusion. A case study is presented, and strategies for improving patient outcomes are discussed.

Aged↗
PubMed1995

Protamine use during peripheral vascular surgery: a prospective randomized trial.

PURPOSE: One hundred twenty patients undergoing aortic reconstruction (40), infrainguinal bypass (49), and carotid endarterectomy (31) were prospectively enrolled into a double-blind randomized trial to investigate the utility of routine heparin reversal with protamine. METHODS: All patients underwent systemic heparinization with 90 U/kg body weight during operation and after revascularization were randomized to receive either protamine or saline solution for heparin reversal. Blood loss was measured throughout the surgical procedure, and indexes of coagulation and the requirement for blood and blood products were documented during operation and the first 24 hours after operation. RESULTS: Plasma heparin concentration, partial thromboplastin time, and activated clotting time were significantly higher (p < 0.05) in those receiving saline solution at 20 minutes and 1 hour after administration. Total surgical blood loss was not significantly different between study groups. No significant differences were found in blood product requirement, intravenous fluid administered, hematocrit, or wound hematomas between groups at 24 hours. In addition, no difference was seen in the surgeon's subjective intraoperative assessment of hemostasis after administration of either study drug. Furthermore, after study drug administration protamine was associated with a deleterious effect on subsequent intraoperative blood loss (318 +/- 33 ml vs 195 +/- 18 ml, p < 0.05). CONCLUSIONS: Although protamine effectively reverses heparin anticoagulation, its routine use after elective peripheral vascular surgical reconstruction does not appear to provide any clinical benefit.

Aged↗
PubMed1994

Heparin and protamine use in peripheral vascular surgery: a comparison between surgeons of the Society for Vascular Surgery and the European Society for Vascular Surgery.

It was the intent of this study to document, in general, the patterns and complications of heparin and protamine usage during carotid endarterectomy, aortic and femoral-popliteal-tibial reconstructions for occlusive disease, elective and emergent abdominal aortic aneurysmectomy, thromboembolectomy, and dialysis arteriovenous (AV) fistula placement by surgeons from North America and Europe. All vascular surgeons from the Society for Vascular Surgery (SVS) and the European Society for Vascular Surgery (ESVS) were surveyed by a voluntary, self-reported questionnaire. Six hundred and forty-six completed questionnaires (284 from SVS and 362 from ESVS), representing a 62% response rate, were returned for evaluation. Systemic and regional administration of heparin was common during vascular procedures performed by both SVS and ESVS surgeons. Use of protamine to reverse heparin anticoagulation varied among SVS and ESVS surgeons, respectively, during: carotid endarterectomy (54% vs. 26%, p < 0.01), elective aortic reconstruction for occlusive disease (58% vs. 23%, p < 0.001), elective aortic reconstruction for abdominal aortic aneurysm (63% vs. 27%, p < 0.001), and femoral-popliteal-tibial reconstruction (44% vs. 15%, p < 0.001). Adverse reactions to protamine among the 25,219 and 12,902 cases reported from SVS and ESVS surgeons, respectively, included: hypotension (1209 and 495 cases), pulmonary artery hypertension (65 and eight cases), anaphylaxis (52 and 10 cases), and death (seven and two cases). These adverse responses accounted for 5.3% and 4.0% of the SVS and ESVS cases, respectively. Although this study is subject to the known limitations of a retrospective survey, it is clear that heparin use is common. Protamine reversal of heparin anticoagulation is more common in North America.(ABSTRACT TRUNCATED AT 250 WORDS)

Data Collection↗
PubMed1999

The use of low-dose heparin is safe in carotid endarterectomy and avoids the use of protamine sulfate.

Controversy exists concerning the appropriate dose of heparin needed during carotid endarterectomy. Use of high-dose heparin (100 U/kg) during carotid endarterectomy may require the use of protamine to minimize perioperative bleeding complications. At the authors' institution the use of 30 U/kg heparin for arterial reconstruction has obviated the need for protamine. A retrospective study of carotid endarterectomies performed was undertaken. Patients undergoing combined procedures with carotid endarterectomy were excluded. A total of 420 carotid endarterectomies were performed in 330 patients. All received 3000 U of heparin or less during carotid endarterectomy. Non-fatal stroke and transient neurological deficits occurred in 0.48% and 1.9%, respectively. Mortality was 0.9%. Wounds were dry in 97%, swollen in 2.5% and bloody in 0.5%. No patient received protamine. Two patients were returned to the operating room for re-exploration because of hematoma. In conclusion, the use of protamine may be safely avoided with 30 U/kg heparin, and give acceptable stroke- and minimal complication rates.

Anticoagulants↗
PubMed1996

Protamine-induced fulminating non-cardiogenic pulmonary edema following cardiopulmonary bypass.

Although fulminating non-cardiogenic pulmonary edema following cardiopulmonary bypass is not a new phenomenon, the exact cause and pathogenesis are still unknown. The causative agent of this potentially lethal syndrome was hypothesized to be either protamine or blood products. There was no evidence that an antibody-mediated reaction plays a role and patients were reported to have negative skin tests. We report, for the first time, a patient who presented with fulminating non-cardiogenic pulmonary edema after repeated protamine administration with a subsequent positive skin test to protamine. Specific IgG antibodies to protamine were also found; however, the level was in the range of that of exposed non-reacting controls. IgE antibodies could not be detected.

Aortic Aneurysm↗
PubMed1996

Age-related differences in heparin sensitivity and heparin-protamine interactions in cardiac surgery patients.

OBJECTIVE: The present study was conducted to determine how children and adults differ (it at all) with respect to sensitivity to heparin activity and heparin-protamine interactions during cardiac surgery requiring cardiopulmonary bypass (CPB). DESIGN: A prospective study of both children and adults undergoing CPB. SETTING: A tertiary care academic medical center between July 1992 and October 1994. PARTICIPANTS: Ninety patients who had cardiac or aortic arch surgery using CPB. The median age of the entire study sample was 15.8 years (range 2 months to 72 years). INTERVENTION: Data were obtained using the Medtronic Hemotec Hepcon Hemostasis Management System (Englewood, CO). An ex vivo heparin dose-response (HDR) curve was generated for each patient before skin incision to determine the target heparin concentration (THC) needed to achieve an activated coagulation time (ACT) of at least 480 seconds. Protamine dose was determined on the basis of whole blood heparin concentration estimated by means of a heparin-protamine titration. MEASUREMENTS AND MAIN RESULTS: The study population was divided into four groups based on age: infants (< 1 year), preschool (1 to 5 years), school-age (5 to 14 years) and adults (> 14 years). The mean +/- SD THC for the preschool group was 4.0 +/- 1.1; for infants, 3.3 +/- 0.7; for school-age, 3.1 +/- 0.7; and for adults, 3.4 +/- 0.7. The initial dose of heparin needed to achieve this THC (mean +/- SD) was significantly higher in infants (578 +/- 220 U/kg) and preschool children (477 +/- 159 U/kg) than in school-age children (327 +/- 57 U/kg) and adults (332 +/- 64 U/kg). The ratio of protamine to heparin was significantly higher in adults (1.4 +/- 0.5) and school-age children (1.3 +/- 0.6) than in infants (1.1 +/- 0.7) and preschool children (1.1 +/- 0.4). CONCLUSIONS: Pre-school children are less sensitive to heparin but also display a wider range of sensitivity. The data in this study support the use of 300 U/kg of heparin before CPB in patients > or = 5 years but suggest that heparin requirements may be greater in the younger patient who may require as much as 500 U/kg to achieve what is believed to be an appropriate target heparin concentration for initiating CPB.

Adolescent↗
PubMed1999

Do anticoagulation with heparin and protamine reversal alter thrombogenicity of coated and noncoated pulmonary artery catheters?

OBJECTIVE: To clarify the role of anticoagulation with heparin and protamine reversal on the effectiveness of heparin bonding or coating of pulmonary artery catheters in preventing thrombus formation in the Rhesus monkey. DESIGN: A controlled, unblinded, open-labeled study. SETTING: A research laboratory at Tulane School of Medicine (New Orleans, LA). PARTICIPANTS: Twenty-four anesthetized Rhesus monkeys. INTERVENTIONS: The monkeys were assigned to one of four groups. In the first group (group A), non-heparin-coated catheters were inserted into a femoral vein through an incision, advanced proximally for a distance of 30 cm, and left in place for 1 hour. In the second group (group B), heparin-coated catheters were placed and evaluated in the same manner as in group A. In the third group (group C), the primates received 3 mg/kg of heparin intravenously (i.v.) before insertion of a non-heparin-coated pulmonary artery catheter. After the catheter had been in place for 1 hour, protamine, 3 mg/kg i.v., was administered, and the catheter was left in place for an additional hour. In the final group (group D), the primates received 3 mg/kg of heparin i.v. before insertion of a heparin-coated pulmonary artery catheter. After the catheter had been in place for 1 hour, protamine, 3 mg/kg i.v., was administered, and the catheter was left in place for an additional hour. In each group, the catheter was withdrawn with the balloon inflated to minimize any stripping of thrombus from the surface of the catheter. Thrombus was removed from the catheter through a femoral vein cutdown and weighed. Laboratory values were determined for each animal, and clot formation was evaluated in each group. After completion of the study, the animals were returned to the primate breeding colony. MEASUREMENTS AND MAIN RESULTS: There were no significant differences in hematocrit, prothrombin time, partial thromboplastin time, or platelet levels among the four groups; therefore, the data were pooled. Clots were observed on five of six catheters in both groups A and C; however, clot formation was seen in one of six catheters in group B and three of six catheters in group D. There was a statistically significant difference (p < 0.01) in mean clot weight in group A (265 +/- 68 mg; range, 0 to 447 mg) compared with 13 +/- 13 mg in group B (range, 0 to 80 mg). There was no significant difference in mean clot weight in group C (104 +/- 35 mg; range, 0 to 202 mg) compared with group D (24 +/- 16 mg, range, 0 to 98 mg). Additionally, in group C, the mean clot weight was significantly less than in group A. CONCLUSION: Anticoagulation of primates with heparin before catheter insertion imparts some protection to non-coated catheters, and protamine reversal of anticoagulation with heparin may partially negate the protective effect seen with heparin-coated pulmonary artery catheters.

Animals↗
PubMedDate not supplied

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells.

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.

Active Transport, Cell Nucleus↗
PubMed2002

Nucleoplasmin interaction with protamines. Involvement of the polyglutamic tract.

Different recombinant forms of nucleoplasmin including truncations at the carboxyl-terminal end of the molecule (r-NP121, r-NP142) have been expressed and purified. All of them were found to oligomerize, forming pentameric complexes which, according to their hydrodynamic properties, can be modeled by oblate ellipsoids of constant width. In this model, the highly charged carboxyl ends appear to be arranged around a pentameric core along the plane defined by the major axes of the ellipsoid. Importantly, all the recombinant forms appear to be able to decondense protamine-containing sperm nuclei. However, although the stoichiometry with which protamines bind to these forms appears to be constant (2.5 mol of protamine/mol of nucleoplasmin pentamer), the efficiency with which they remove protamines from the sperm DNA decreases in the following order: o-NP > r-NP142 > or = r-NP >> r-NP121. Therefore, the main polyglutamic tract of nucleoplasmin (which is absent in r-NP121), while enhancing the efficiency of protamine removal, is not indispensable for sperm chromatin decondensation in the biological model we have used.

Amino Acid Sequence↗
PubMed1997

Analysis of DNA-protamine interactions by optical detection of magnetic resonance.

Optically detected magnetic resonance (ODMR) has been used to identify the binding site of a synthetic protamine subdomain to the major groove of DNA. A 14 amino acid peptide (R6WGR6) analog of the central DNA binding domain of bull protamine was synthesized with phenylalanine replaced by tryptophan (Trp). The peptide was bound to double-stranded poly(dABrdU) and to calf thymus DNA (CT DNA) and the complexes characterized as "wet" solids using ODMR techniques. The appearance of the D + E transition in the slow passage ODMR and of short-lived components in the phosphorescence decay of the complex of R6WGR6 with poly(dABrdU) is diagnostic of a heavy atom effect. This can only occur if the peptide binds in the major groove of poly(dABrdU). The microenvironment of Trp in the nucleoprotein complex was characterized by phosphorescence, radiative decay lifetimes, and low-temperature ODMR measurements before and after binding to DNA. Bathochromic shifts in the phosphorescence emission upon exciting to the red in CT DNA-peptide suggest that the Trp is in a polar environment, while the red-shifted position of the 0, 0-band emission points to a more polarizable environment. The heavy atom effect strongly suggests a Trp location within the major groove of DNA. A partial stacking of Trp with the polarizable nucleobases and simultaneous interactions with the phosphate-guanidinium ion pairs and/or water molecules in the major groove of DNA which might not be totally displaced upon binding of the peptide could explain this conflicting evidence. Extrapolation of results from the system studied to protamine binding in sperm chromatin strongly suggests that the predominant binding site of protamine is the major groove of DNA.

Amino Acid Sequence↗
PubMedDate not supplied

Cation-exchange displacement chromatography of proteins with protamine displacers: effect of induced salt gradients.

Protamine was investigated for its utility as a protein displacer in cation-exchange systems. Although the protamine solution contained several variants of the molecule, the high affinity of all of the components in this heterogeneous biopolymer enabled it to act as an efficient protein displacer. To facilitate parameter estimation of the protamine, a preliminary purification was carried out by preparative elution chromatography. Chromatographic parameters of both the feed proteins and protamine displacer were obtained for use in a multicomponent steric mass action ion-exchange displacement model. Model simulations were compared to displacement results under both moderate and intense induced salt gradient conditions. In both cases, excellent agreement was obtained between the displacement experiments and theoretical predictions. In addition, these studies serve to dramatize the importance of induced salt gradients in ion-exchange displacement systems.

Amino Acid Sequence↗
PubMed2003

Voltammetry of the phase transfer of polypeptide protamines across polarized liquid/liquid interfaces.

Using natural polypeptide protamines as the illustrating example, we observed the phase transfer of biological polyions at polarized interfaces between two immiscible electrolyte solutions (ITIES) for the first time by micropipet voltammetry. Analysis of the limiting currents indicates that each protamine carries 20 positive charges as expected from the number of arginine residues in the molecule. Also, it was shown that the shape of the voltammograms is limited by the interfacial transfer of protamines. Coupling of the transfer reactions with interfacial adsorption/desorption of protamines was also observed. ITIES will serve as a basis of amperometric sensors for biological macromolecules and also as a model for understanding potential-driven membrane transport of these molecules.

Electrochemistry↗
PubMed1995

Squid spermiogenesis: molecular characterization of testis-specific pro-protamines.

Cuttlefish spermiogenesis is characterized by a two-step nuclear protein transition: histones-->spermatid-specific protein (protein T)-->sperm protamine (protein Sp). A similar situation can be observed in another Cephalopod species, the squid Loligo pealeii. The protein T from Loligo consists of two structural variants, T1 and T2 (molecular masses: 10788 and 10791 Da respectively), phosphorylated to different degrees (2-6 phosphate groups). The primary structures of these two variants and of the protamine variant Sp2 were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are closely related proteins of 79 residues. The complete structural identity of the C-terminal domain (residues 22-79) of protein T2 with the sperm protamine Sp2 (molecular mass 8562 Da, 58 residues) strongly suggests that the testis-specific protein T2 is indeed the precursor of the protamine. The transition between the precursor protein T and protein Sp results from a hydrolytic cleavage similar to that found in many proteins that are synthesized as precursors. The processing mechanism involves the specific cleavage of a Gly-Arg bond in the sequence Met/Leu18-Lys-Gly-Gly-Arg-Arg23. Furthermore, the study provides molecular evidence on the taxonomic relationship between Loligo and Sepia.

Amino Acid Sequence↗