Purification and characterization of particulate guanylate cyclase from sea urchin spermatozoa

The particulate form of guanylate cyclase from sea urchin spermatozoa was purified to apparent homogeneity by chromatography on GTP-Sepharose and DEAE-Sepharose and by preparative gel electrophoresis. The sedimentation coefficient (S20,w) was 6.8 and the Stokes radius was 5.1 nm, from which a native molecular weight of 157,000 was calculated. A single protein or periodic acid-Schiff staining band of 135,000 Da was observed after Na dodecyl SO4 gel electrophoresis. Antibody was produced to guanylate cyclase and was shown by electrophoretic transfer experiments (Western blot) to interact with only the Mr = 135,000 band in cases where all of the detergent-extracted protein from spermatozoa was added to the Na dodecyl SO4 gels. Although guanylate cyclase was normally bound to concanavalin A-Sepharose, after endoglycosidase H treatment it failed to bind. Treatment of the enzyme with endoglycosidase H did not alter guanylate cyclase activity, but the apparent size of the enzyme decreased to 72,000 Da on Na dodecyl SO4 gels. An analysis of carbohydrate composition indicated that the oligosaccharides contained N-acetylglucosamine, mannose, galactose, and 2-aminoerythritol in molar ratios (1:3:0.75:2); after endoglycosidase H treatment the enzyme contained essentially no carbohydrate. Major amino acids in the enzyme were aspartic (Asn) and glutamic (Gln) which accounted for approximately 25 mol % of the enzyme amino acid composition. The purified enzyme displayed linear kinetics on double reciprocal plots and had a KMnGTP = 133 microM, KM2+ = 138 microM, KiMnGTP = 122 microM, KiMn2+ = 127 microM, and a V max in excess of 15 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C. Sodium nitroprusside did not stimulate the enzyme in either the presence or absence of added hemeproteins. These results indicate that the particulate form of guanylate cyclase from sea urchin spermatozoa is a glycoprotein which is distinctly different than the soluble form of the enzyme found in mammalian tissues.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

A soluble adenylyl cyclase from sea urchin spermatozoa

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

Purification and characterization of the sea urchin embryo hatching enzyme

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.     

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

• 1.1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively.
• 2.2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The K_m value for p-nitrophenyl sulfate was 0.59 mM.
• 3.3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.

     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Purification and characterization of trypsin-like enzyme from sea urchin eggs: substrate specificity and physiological role

A trypsin-like enzyme has been purified to homogeneity from eggs of the sea urchin, Strongylocentrotus intermedius. The purified enzyme efficiently hydrolyzed Z-Phe-Arg-4- methylcoumaryl -7-amide (MCA) and Pro-Phe-Arg-MCA among 12 peptidyl-Arg (or Lys)- MCAs . The substrate specificity of the enzyme was closely similar to that of the enzyme activity in the egg cortical granule exudate. Among various peptidyl-argininal (Arg-H) derivatives, Z-Phe-Arg-H and Z-Phe-Leu-Arg-H showed the strongest inhibition against both the activity of the purified enzyme and the elevation of vitelline coat. Thus, the trypsin-like enzyme of sea urchin possesses a narrow substrate specificity and participates at least in the elevation of vitelline coat during fertilization.     

Purification and properties of soluble actin from sea urchin eggs

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.     

Inhibition by palmitoyl CoA of dynein ATPase from sea urchin spermatozoa

ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 microns, and was stimulated at concentrations between 2 microns and 10 microns. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1 mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF1 ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.     

Purification and characterization of Contractin A from the pedicellarial venom of sea urchin, Toxopneustes pileolus

A component that causes contraction of the isolated guinea pig tracheal smooth muscle was isolated in homogeneous form from the venom of the pedicellaria of the sea urchin, Toxopneustes pileolus. It is named Contractin A. Contractin A has 18,000 Da with a total residue of 138 amino acids. The molecular weight is about 17,700. The N-terminal amino acid is serine. The partial amino acid sequence was determined up to 37 residues. Direct comparison of sea urchin Contractin A does not show any similarity in amino acid sequence to toxins isolated from other marine toxin producers such as sea snakes, sea anemones, or marine worms. Contractin A caused contraction of the tracheal smooth muscle in a dose-dependent manner. Furthermore, Contractin A relaxed the contraction induced by histamine. The contraction and relaxation activity of Contractin A on the tracheal smooth muscle is reduced by a cyclooxygenase inhibitor such as indomethacin. The contraction induced by Contractin A is also inhibited by a phospholipase C inhibitor but not by a phospholipase A2 inhibitor. These results suggest that in the isolated guinea pig tracheal smooth muscle, the response to Contractin A may be effected through activated phospholipase C.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.     

Purification and characterization of calmodulin from rat liver mitochondria

Mitochondrial calmodulin of rat liver was purified and classified. It co-migrated with bovine brain calmodulin in non-denaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis and isoelectric focusing. The mitochondrial calmodulin activated Ca²⁺-dependent phosphodiesterase of bovine brain in the presence of Ca²⁺. About 80% of the mitochondrial calmodulin was proved to be of cytosol origin. It was easily detached by washing with buffer containing EGTA. The other 20% was intramitochondrial calmodulin; half of it was in the matrix space, and half in the membrane.     

Purification and some properties of ATP-sulfurylase from developing sea urchin embryos

ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4), purified about 200-fold from sea urchin embryos, was free of ATPase and inorganic pyrophosphatase. The molecular weight of the enzyme was approx. 280 000 measured by gel filtration. The enzyme was activated by Mg2+, Ca2+ or Zn2+; EDTA and p-chloromercuriphenylsulfonate inhibited the enzyme activity. The inhibition was reversed by addition of Mg2+ and dithiothreitol, respectively. The enzyme activity increased continuously as the pH was raised from 5.6 to 10.6. The Km values for the enzyme were calculated to be 13 microM for adenosine 5'-phosphosulfate and 23 microM for pyrophosphate.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.