Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) in sea urchin embryos

Sea urchin embryos were labeled with [3H]adenosine at two developmental stages (morula and prism) and the labeled acid-soluble nucleotides were fractionated successively by column chromatography with DEAE-Sephadex and DEAE-cellulose, and by thin-layer chromatography on a PEI-cellulose plate. Significant radioactivity was detected on the PEI-cellulose plate at the region of diadenosine 5',5'-P1,P4-tetraphosphate (AP4A). After treatment of this fraction with phosphodiesterase, the radioactivity was all recovered in the AMP region, while alkaline phosphatase had no effect on the AP4A fraction. The present result suggests that AP4A is actively synthesized in the sea urchin embryos.     

Nuclear AP4A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

The AP4A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). Among various subcellular components that can bind [3H]AP4A, nuclei alone showed the highly specific Ap4A-binding activity which was not influenced by the presence of AP4A, AP5A and GP4G. The addition of an excess amount of ATP only slightly reduced the binding of [3H]AP4A to the nuclei. It was found that AP4A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP4A-binding activity fluctuated cyclically during each cell cycle, with a transient increase at the beginning of S phase followed by an abrupt decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP4A-binding activity was also prevented. It seems that the binding of AP4A to the nuclear structural protein is involved in the initiation of S phase.     

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.     

Partial characterization of a nuclear proteolytic activity from fertilized sea urchin eggs

The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase in the sea urchin embryo is developmentally regulated

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, an enzyme which plays a regulatory role in the synthesis of cholesterol, dolichol, and coenzyme Q, has been measured in the developing embryo of the sea urchin. Enzyme activity increased at least 200-fold during development from the unfertilized egg to the pluteus stage embryo. Mixing experiments suggested that the low level of enzyme activity found at early stages was not due to the presence of inhibitor(s) in the egg or zygote. The enzyme in the sea urchin embryo exhibited properties different from that found in mammals: only a fraction of the activity could be solubilized from microsomes, and mild trypsinization inactivated the enzyme without releasing any of it from the microsomes in soluble form. To further study the sea urchin HMG-CoA reductase, a genomic clone was identified by hybridization to a cDNA encoding hamster HMG-CoA reductase. Sequence analysis of this clone revealed a coding region that shares a high degree of homology with the carboxyl-terminal domain of hamster HMG-CoA reductase. Analysis of sea urchin embryo HMG-CoA reductase mRNA levels using a restriction fragment derived from the genomic clone revealed a 5.5-kilobase poly(A)+ mRNA that increased 15-fold during development from the egg to the gastrula stage and then decreased 1.5-fold at the pluteus stage. Since the relative increase in HMG-CoA reductase mRNA was less than the increase in enzyme activity (15-fold versus 200-fold) factors in addition to the level of mRNA may control the activity of this enzyme during embryogenesis.     

CHANGE IN THE ACTIVITY OF ARYLESTERASE IN SEA URCHIN EGGS FOLLOWING FERTILIZATION

The activity of arylcsterase in sea urchin eggs ( Anthocidaris craxsispina ), increases at 5 min after fertilization to about 1.5-fold that in unfertilized eggs, and decreases at 15 min to a lower level than that in unfertilized eggs. Then the activity of the enzyme increases again. The enzyme activity in unfertilized eggs is enhanced by either fructose 1, 6-diphosphate (FDP) at concentrations between 4 and 10 μM, or guanosine 3', 5'-cyclic monophosphalc (cGMP) at concentrations between 0.1 and 0.3 μM. The activity is detectable in the crude microsomal fraction and also in the supernatant fraction obtained from sea urchin egg homogenates by centrifugation at 105,000 × g for 2 hr.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

SALT-DEPENDENT PROPERTIES OF HATCHING ENZYME FROM EMBRYOS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS

The effect of salts on hatching enzyme and protease from the embryo of the sea urchin, Hemicentrotus pulcherrimus , was investigated. The culture medium containing hatching enzyme secreted from the hatched blastula was dialyzed against Tris-HCl (pH 8.0) with or without salts. Both hatching enzyme and protease were activated and stabilized by CaCL₂, NaCI and KCI, while inhibited by MgCI₂. Protease activity was maximal at about 0.25 M NaCI. KCI, NH₄, CI and LiCI. Maximal activity of hatching enzyme was obtained at 0.5 M NaCl, KCI and NH₄ CI, while activity was inhibited by any concentration of LiC1. Among monovalcnt cations, the order of activation was NaCI, KCI > NH₄Cl. The activity of hatching enzyme was stabilized by dialysis against 1 M NaCI or KCI in the presence of CaCl.₂, but was rapidly lost by dialysis against lower concentrations of salts. Reactivation of hatching enzyme was not achieved by redialysis against I M NaCI. On the other hand, protease was reactivated by I M NaCI or KCI. From these results, hatching enzyme of the sea urchin may be called a moderate halophilic enzyme. It was assumed that at least two enzymes exist in the crude enzyme preparation and that they may have different functions.     

Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, alpha-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymotrypsin."     

Adenylate cyclase from sea urchin eggs is positively and negatively regulated by D-1 and D-2 dopamine receptors

The adenylate cyclase present in membranes prepared from sea urchin eggs is sensitive to dopamine stimulation. The receptor sites coupled to sea urchin adenylate cyclase were characterized by means of specific agonists and antagonists. The D-1 dopamine agonist SKF-38393 was able to stimulate enzyme activity, while the two D-1 dopamine antagonists, SCH-23390 and SKF-83566, suppressed the stimulatory effect of dopamine. In addition, the D-2 dopamine agonists, PPHT and metergoline, brought about a dose-dependent inhibition of dopamine-stimulated adenylate cyclase activity. These data show that: (i) in sea urchin eggs adenylate cyclase is regulated by dopamine receptors; (ii) these receptors share characteristics with D-1 and D-2 dopamine receptors present in the mammalian brain.     

Effect of Ca2+, Mg2+-dependent deoxyribonuclease on DNA synthesis in cell nuclei from embryos of the sea urchin Strongylocentrotus intermedius

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.     

Soluble epoxide hydrolase homologs in Strongylocentrotus purpuratus suggest a gene duplication event and subsequent divergence

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.     

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.     

Novel salt-resistant alkaline phosphatase from eggs of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

It was shown that the alkaline phosphatase (AP) isolated from eggs of the sea urchin Strongylocentrotus intermedius was a homodimer with a molecular mass of 150 kDa and that it exhibited maximal activity at a рН of 8.1–8.5 and a temperature of 45°С. Mg²⁺, Ca²⁺, and Mn²⁺ ions, as well as DTT, are activators of AP activity, while Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, and Ni²⁺ ions, as well as EDTA, N-EMI, and p-CMB, inhibit its activity. The enzyme demonstrates unique salt resistance and can hydrolyze the substrate in seawater. It was shown by fluorescence methods and circular dichroism that an increase of the NaCl concentration above 1.0М caused noticeable changes in the secondary and tertiary structures of the protein and a decrease in enzyme activity. Analysis of the molecular masses of tryptic peptides conducted by mass spectrometry with the MASCOT program (which is based on the NCBI and SWISS-PROT databases) did not allow identification of the investigated protein. The uniqueness of the source and of the properties of the investigated AP, which were not characteristic for nonspecific AP, as well as the difficulties in primary structure identification with the fingerprinting technique, suggested that this enzyme was a nontypical AP with a novel structure.     

The action of Laminaria japonica extracts on 1,3-beta-D-glucanase, a digestive enzyme of the Strongylocentrotus intermedius sea urchin

Nutritional attractiveness of the brown alga Laminaria japonica for the sea urchin Strongylocentrotus intermedius was studied. The composition of L. japonica was analyzed after one and two years of its life under natural conditions, in its seedlings, and in the alga partially degraded by natural factors. Substances extracted with various solvents were tested for the presence of inhibitors and activators of 1,3-beta-D-glucanase, a digestive enzyme of the sea urchin. Ethanolic extract of freshly harvested L. japonica was found to suppress the enzyme activity. Substances present in ethanolic extracts of the alga after one or two years of its life cycle and in the alga, partly degraded by natural factors, activated the sea urchin enzyme. This fact is in agreement with earlier natural observations concerning the nutritional attractiveness of such L. japonica samples for Strongylocentrotus intermedius.     

Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

Role of dopamine and indolamine derivatives in the regulation of the sea urchin adenylate cyclase

The adenylate cyclase of the sea urchin egg is stimulated by dopamine in the presence of GTP. The enzyme activity is strongly enhanced when Gpp (NH)p is substituted for GTP, or after cholera toxin treatment. Gramine, an indolamine derivative, brings about non-competitive inhibition of the dopamine-stimulated adenylate cyclase activity. Pertussis toxin causes an attenuation of the gramine-induced inhibition of adenylate cyclase. These results show that dopamine and indolamine derivatives partecipate in the regulation of the adenylate cyclase activity of the sea urchin egg.     

Rapid partial purification of S-adenosyl-L-methionine decarboxylase by affinity chromatography

A Sepharose-ethylenediamine-PCMB column can be used to obtain a rapid purification of S-adenosyl-L-methionine decarboxylase. PCMB-affinity fractions from both rat liver and sea urchin eggs have high specific activity, particularly the latter. The activity of the purified rat liver enzyme is stimulated by the addition of either putrescine or spermidine, whereas the purified enzyme fraction from sea urchin eggs has no measurable activity without the addition of either putrescine or spermidine. In both preparations there is a stoichiometric relationship between the release of ¹⁴CO2 from S-adenosyl-L-carboxyl-¹⁴C-methionine and the formation of spermidine.