Protamines

Felix, K.

doi:10.1016/s0065-3233(08)60307-1

Cited by 61 publications

(17 citation statements)

References 99 publications

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“…Protamines from spermatozoa of both steelhead trout and coho salmon --were purified as described and found to contain less than 7 per cent of their serine residues in the phosphorylated form ( Table 2). The lack of phosphorylation _IN _----r n -of this material is in agreement with all previous work on protamines,'5 16,18,19 where either milt expressed from the fish or mature testis was used as starting material in the isolation and purification of protamine. In these earlier studies the presence of 0-phosphoserine was not amino acid residues.…”

supporting

confidence: 89%

Phosphorylation of protamine during spermatogenesis in trout testis.

Ingles¹,

Dixon²

1967

Proc. Natl. Acad. Sci. U.S.A.

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Recent experiments by Kleinsmith, Allfrey, and Mirskyl and Stevely and Stockenl2 have clearly demonstrated the incorporation of P32-phosphate into the histones of calf and rat thymus nuclei, the site of linkage of this phosphate being 0-phosphoserinel' 2 or O-phosphothreonine.1 Considerable interest focuses upon these findings as suggesting a means whereby the activity of histones in complexing with DNA may be modulated. Kleinsmith et al.' have reported that an increase in phosphorylation of nuclear proteins correlates with an increase of RNA synthesis in lymphocytes stimulated to grow and divide by phytohemagglutinin, while Stevely and Stocken2 have shown an increased ability of complexes of DNA with phospho-fl-histone to prime in vitro RNA synthesis by RNA polymerase as compared with the nonphosphorylated histone-DNA complex.This paper reports studies on protamines labeled with P32-phosphate or C14-serine by incubation of dissociated intact cells from testes of steelhead trout (Salmo gairdnerii). Development of the testes and spermatogenesis was induced4 by injecting the fish with an extract of pituitary glands from salmon. Testes were obtained in this way conveniently and reproducibly at various stages of maturation. During the later stages of maturation these cells, which are actively synthesizing the characteristic sperm nuclear protein, protamine,3 have been shown to incorporate P32-phosphate into both histone and protamine fractions.The presence of 0-phosphoserine in partial acid hydrolysates of a commercially available preparation of protamine (salmine) was noted by Murray.5 More recently Langan and Smith6 have isolated a kinase from rat liver which catalyzes the phosphorylation of protamine and histones in vitro.In the present work phosphorylation has also been shown to be at serine residues, and several labeled phosphopeptides have been isolated by tryptic digestion of P32-protamine. Parallel incorporation of C14-serine and P32-phosphate into protamine shows that all newly incorporated serine residues are phosphorylated. Since the total content of 0-phosphoserine in protamine falls during the later stages of maturation of the testis, a rapid turnover of phosphate is indicated. This view is confirmed by the almost complete absence of phosphate in protamine extracted from mature sperm collected from naturally spawning trout and salmon.Methods.-Cells at a stage active in protamine biosynthesis were obtained from the testes of steelhead trout in which spermatogenesis had been induced by injections of salmon pituitary extracts.4 Mature spermatozoa were collected by expressing milt from both steelhead trout and coho salmon (Onchorhynchus kisutch) during annual spawning operations at fish hatcheries on the Skagit River (State of Washington Departments of Game and Fisheries).Incubation procedures: Cell suspensions from the developing trout testes were prepared at 0°C as previously described.3 Minced tissue was suspended in buffer (0.14 M NaCl, 5.4 mM KCl, 0.81 mM MgSO4, 1.26 mM CaCl2, 5.6 mM glucose, 4.2 mM Na...

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supporting

confidence: 89%

Phosphorylation of protamine during spermatogenesis in trout testis.

Ingles¹,

Dixon²

1967

Proc. Natl. Acad. Sci. U.S.A.

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“…Protamines are widely used heparin antidotes (Byun et al, 1999), are highly positively charged molecules (Felix, 1960), and are mixtures of closely related basic proteins. In the present study, clupeine was chosen because its composition is well characterized and its three components (YI, YII, and Z) are fully sequenced.…”

Section: Discussionmentioning

confidence: 99%

“…Protamines are a known antidote to heparin (Byun et al, 1999), are highly positively charged, and are rich in basic amino acids (Felix, 1960). These proteins are known to bind tightly to DNA and are used in DNAbinding assays (Raukas and Mikelsaar, 1999).…”

Section: Introductionmentioning

confidence: 99%

Reversible Block of the Calcium Release Channel/Ryanodine Receptor by Protamine, a Heparin Antidote

Koulen

Ehrlich

2000

MBoC

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Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C 2 C 12 mouse muscle cells prevented caffeineinduced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity. INTRODUCTIONRyanodine receptors (calcium-induced calcium release channels; RyR) play a crucial role in most cell types, including muscle cells, neurons, and epithelial cells. They mediate the release of calcium ions from the endoplasmic/sarcoplasmic reticulum into the cytosol and thereby convert a number of extracellular stimuli into intracellular calcium signals. RyRs are large tetrameric proteins that show sequence similarity with inositol 1,4,5-trisphosphate (IP 3 )-gated calcium channels of the endoplasmic/sarcoplasmic reticulum, but they are distinct in their biophysical and pharmacological properties (Smith et al., 1986;Ehrlich and Watras, 1988;Supattapone et al., 1988;Mignery et al., 1989;Palade et al., 1989. For example, highly negatively charged polyanions such as pentosan polysulfate, polyvinyl sulfate, and heparin increased the activity of RyRs, whereas they decrease the activity of IP 3 receptors (Bezprozvanny et al., 1993). The authors of these studies reported that to get an increase in channel open probability, several polyanion molecules needed to bind to the RyR. They provided evidence for a mechanism in which the binding of polyanions to the RyR would increase the local negative charge of the receptor complex and thus attract more calcium ions to activate the receptor. Additional support for this mechanism is found in the facts that the endogenous RyR has a fixed negative surface charge and that this surface charge potentiates conduction and conveys divalent cation selectivity of the channel (Tu et al., 1994).In the present study, we analyzed the effects of protamine (clupeine) on the RyR. Protamines are a known antidote to heparin (Byun et al., 1999), are highly positively charged, and are rich in basic amino acids (Felix, 1960). Thes...

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“…In order to determine if the inhibitory action of heparin on isolated platelet aggregation was related specifically to heparin and not to the impurities in the commercial solutions, heparin was pretreated with heparinase [28] or with protamine sulfate [29,30], following methods previously described, with some modifications. These preincubated solutions were then assayed in a platelet aggregation system, as well as in Thrombin-Clotting Time.…”

Section: Heparin Neutralization Study With Heparinase and Protamine Smentioning

confidence: 99%

Effect of heparin on platelet aggregation

Saba

Morelli

1984

American J Hematol

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The effect of heparin on platelet aggregation was systematically examined on platelets in plasma (PRP), as well as on gel-filtered, washed, and formaldehyde-fixed platelets. Results indicate that, although heparin causes a mild potentiation of platelet aggregation in the PRP systems, a significant inhibitory activity is observed when heparin is added to isolated platelets. This inhibitory activity appears to be specific and not related to the impurities in the heparin preparations, as heparinase, as well as protamine, effectively neutralizes the heparin-mediated inhibitory activity on platelet aggregation. Although heparin-mediated inhibitory activity can be demonstrated in the presence of a number of different agonists (ADP, arachidonic acid, thrombin, Ionophore A23187, epinephrine, and ristocetin), the most pronounced inhibition is seen in the presence of ristocetin. Further studies show that heparin enhances thromboxane generation in isolated platelets. Platelets pretreated with heparin, however, fail to respond to preformed thromboxane. These findings suggest that, in addition to the potentiation of thromboxane production in platelets, heparin may also attribute some change(s) to the platelet(s)/platelet membrane, which interferes with their ability to respond to the agonists of platelet aggregation. This antiaggregatory activity of heparin was found to be inhibited by a factor(s) present in plasma but not in serum.

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Protamines

Cited by 61 publications

References 99 publications

Phosphorylation of protamine during spermatogenesis in trout testis.

Phosphorylation of protamine during spermatogenesis in trout testis.

Reversible Block of the Calcium Release Channel/Ryanodine Receptor by Protamine, a Heparin Antidote

Effect of heparin on platelet aggregation

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