scholarly journals Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein.

1985 ◽  
Vol 100 (3) ◽  
pp. 938-946 ◽  
Author(s):  
P J Weidman ◽  
E S Kay ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Cortical Granules ◽  
Binding Interaction ◽  
Membrane Assembly ◽  
Calcium Dependent ◽  
Structural Subunit ◽  
Fertilization Membrane

Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross-linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase-proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.

Studies on the effects of magnesium ion and propranolol on iris muscle phosphatidate phosphohydrolase

1984 ◽  
Vol 62 (2-3) ◽  
pp. 170-177 ◽  
Author(s):  
Ata A. Abdel-Latif ◽  
Jack P. Smith
Keyword(s):  
Smooth Muscle ◽  
Divalent Cation ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Soluble Enzyme ◽  
Microsomal Enzyme ◽  
Time Intervals ◽  
Phosphatidate Phosphohydrolase

The properties, subcellular distribution, and the effects of Mg2+ and propranolol on phosphatidate phosphohydrolase (EC 3.1.3.4) from rabbit iris smooth muscle have been investigated. The particulate and soluble (0–30% (NH4)2SO4 fraction) enzymes were assayed using aqueous phosphatidate dispersions and membrane-bound phosphatidate as substrates, respectively. When measured with aqueous substrate, activity was detected in both the particulate and soluble fractions, with the highest relative specific activity found in the microsomal fraction. Maximum dephosphorylation by the microsomal enzyme was about 1100 nmol of inorganic phosphate released/h per milligram protein and occurred at pH 7.0–7.5. In general Mg2+ inhibited the phosphohydrolase activity of the microsomal fraction and stimulated that of the soluble fraction, and the effects of the divalent cation on both of these activities were reversed by propranolol. The microsomal enzyme was slightly stimulated by deoxycholate and inhibited by the divalent cations Mg2+, Ca2+, and Mn2+ at concentrations > 0.25 mM. In contrast, the soluble enzyme was stimulated by Mg2+. Inhibition of the microsomal enzyme by Mg2+ (0.5 mM) was reversed by both EDTA, which also stimulated at higher concentrations (1 mM), and propranolol (0.1–0.2 mM). The inhibitory effect of Ca2+ on the enzyme was not reversed by propranolol. In the absence of Mg2+, the microsomal enzyme was inhibited by propranolol in a dose-dependent manner, and both in the absence and presence of the divalent cation the soluble enzyme was inhibited by the drug in a similar manner. These data suggest that the cationic moiety of propranolol may act by competing at the Mg2+-binding sites. Addition of propranolol (0.2 mM) to iris muscle prelabelled with [14C]arachidonic acid increased accumulation of [14C]phosphatidic acid at all time intervals (2.5–90 min) and brought about a corresponding initial decrease in the formation of [14C]diacylglycerol at short time intervals (2.5 min), thus implicating the phosphohydrolase as a possible site of action of the drug on glycerolipid metabolism in this tissue. In addition to reporting on the characteristics and distribution of phosphatidate phosphohydrolase in the iris smooth muscle, the data presented add further support to our hypothesis that propranolol redirects glycerolipid metabolism in the iris by exerting multiple effects on the enzymes involved in their biosynthesis.

scholarly journals THE ISOLATION OF A MAJOR STRUCTURAL ELEMENT OF THE SEA URCHIN FERTILIZATION MEMBRANE

1970 ◽  
Vol 44 (3) ◽  
pp. 635-644 ◽  
Author(s):  
Joseph Bryan
Keyword(s):  
Sea Urchin ◽  
Butyric Acid ◽  
Strongylocentrotus Purpuratus ◽  
Crystalline Material ◽  
Cortical Granules ◽  
Structural Element ◽  
Fertilization Membrane ◽  
Vitelline Coat

Procedures for isolating the contents of the cortical granules from the ova of the sea urchin, Strongylocentrotus purpuratus, are reported. Dithiothreitol is used to remove the vitelline coat; the "demembranated" eggs are then subsequently activated with butyric acid. By means of these procedures, the hyaline protein and crystalline or paracrystalline material have been isolated from the cortical granules. The crystalline material consists of sheets of cylinders or tubules 150–200 A in diameter. This material is believed to be a major structural element of the fertilization membrane which, in the absence of the vitelline coat, does not form.

scholarly journals ON THE RECONSTITUTION OF THE CRYSTALLINE COMPONENTS OF THE SEA URCHIN FERTILIZATION MEMBRANE

1970 ◽  
Vol 45 (3) ◽  
pp. 606-614 ◽  
Author(s):  
Joseph Bryan
Keyword(s):  
Ionic Strength ◽  
Sea Urchin ◽  
Divalent Cations ◽  
Reducing Agents ◽  
Alkaline Ph ◽  
Mitotic Apparatus ◽  
Fertilization Membrane ◽  
Crystalline Component ◽  
Tubular Components

The characteristics of the reconstitution of a crystalline component of the sea urchin fertilization membrane are presented. The reassembly of large aggregates of cylindrical or tubular components is effected by the addition of calcium or other divalent cations. The reassembly requires a slightly alkaline pH and is little affected by increasing ionic strength. Reassembly is strongly inhibited by treatment with reducing agents such as dithiothreitol. The role of this protein in the formation of the fertilization membrane and its possible relation to the calcium-insoluble proteins of the mitotic apparatus are discussed.

scholarly journals Metabolic similarities between fertilization and phagocytosis. Conservation of a peroxidatic mechanism.

1979 ◽  
Vol 149 (4) ◽  
pp. 938-953 ◽  
Author(s):  
S J Klebanoff ◽  
C A Foerder ◽  
E M Eddy ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Polymorphonuclear Leukocytes ◽  
Cortical Granules ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Hatching Process ◽  
Fertilization Membrane ◽  
Catalyzed Reactions ◽  
Kinetics Of ◽  
Estrogen Binding

At the time of fertilization, sea urchin eggs release a peroxidase which, together with H2O2 generated by a respiratory burst, is responsible for hardening of the fertilization membrane. We demonstrate here that the ovoperoxidase of unfertilized eggs is located in cortical granules and, after fertilization, is concentrated in the fertilization membrane. Fertilization of sea urchin eggs or their parthenogenetic activation with the ionophor A23187 also results in (a) the conversion of iodide to a trichloroacetic acid-precipitable form (iodination), (b) the deiodination of eggs exogenously labeled with myeloperoxidase and H2O2, (c) the degradation of thyroxine as measured by the recovery of the released radioiodine at the origin and in the inorganic iodide spot on paper chromatography, and (d) the conversion of estradiol to an alcohol-precipitable form (estrogen binding). The iodination reaction and the binding of estradio occurs predominantly in the fertilization membrane where the ovoperoxidase is concentrated. From the estimation of the kinetics of incorporation of iodine, we determine that the peroxidative system is active for 30 min after fertilization, long after hardening of the fertilization membrane is complete. Most of the bound iodine is lost during the hatching process. Iodination of albumin is catalyzed by the material released from the egg during fertilization, when combined with H2O2 and iodide. Iodination, thyroxine degradation, and estradiol binding are inhibited by azide, cyanide, aminotriazole, methimazole, ascorbic acid and ergothioneine, all of which can inhibit peroxidase-catalyzed reactions. These responses of the sea urchin egg to fertilization are strikingly similar to the changes induced in polymorphonuclear leukocytes by phagocytosis and, in both instances, a peroxidative mechanism may be involved.

Fertilization Membranes and Sea Urchin Embryos Studied by Scanning Electron Microscopy

1971 ◽  
Vol 29 ◽  
pp. 530-531
Author(s):  
Walter J. Humphreys ◽  
David T. Lindsay
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Sea Urchin ◽  
Structural Features ◽  
Vitelline Membrane ◽  
Cortical Granules ◽  
Freeze Dried ◽  
Fertilization Membrane ◽  
Ultrathin Sections ◽  
Scanning Electron

Scanning electron microscopy (SEM) of specimens freeze-dried after fixation in Parducz fixative and ultrathin sections of the sea urchin, Strongylocentrotus purpuratus show that the egg is covered by many papillae about 0.25μ in diameter and 0.5μ long (Fig. 1a). When the vitelline layer lifts away from the surface of the egg at the time of fertilization it has many uniformly spaced protrusions that persist as prominent and consistent structural features of the fertilization membrane, which forms when material from ruptured cortical granules is added to the inner surface of the raised vitelline membrane. Dimensions of the protrusions, their spacing on the membrane, and their projection in a direction outward from the egg (Fig. 1b) suggests that they originate when the vitelline layer lifts away from the egg surface in the form of a somewhat distorted and expanded replica of the papillae-bearing surface of the unfertilized egg.

scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

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