Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

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.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Isolation and various properties of beta DNA polymerase from the embryos of the sea urchin Strongylocentrotus intermedius

DNA-polymerase beta was isolated from embryonic cells of the sea urchin S. intermedius and purified 1040-fold. The molecular weight of the enzyme is 40 000, sedimentation coefficient 3.2S, pI 8.5. The SH-reagent--N-ethylmaleimide--has no appreciable influence on the enzyme activity. The enzyme is thermolabile and needs four deoxyribonucleoside triphosphates, bivalent metal ions (Mg2+ or Mn2+) and primer template for its activity. The maximal activity is observed when a synthetic polymer--poly(dA).oligo(dT) is used. DNA-polymerase performs DNA synthesis via a distributive mechanism. In terms of physico-chemical properties, the enzyme can be related to DNA-polymerases beta.     

Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis)

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+- ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.     

Water-soluble Ca2+-calmodulin-binding proteins in embryo of the sea urchin Strongylocentrotus intermedius

• 1.1. About 0.3–0.4% of total water-soluble protein extracted from sea urchin embryos at the two-cell and early-gastrula stages was Ca²⁺-dependently bound to immobilized calmodulin.
• 2.2. SDS-PAGE of calmodulin-binding proteins revealed at least 20 polypeptides ranging from 200 to 15.5 kDa, and 70–80% of the protein belonged to a dozen major polypeptides. Polypeptides of 70, 55, 50, 45 and 18 kDa seemed to be the same as those that were detected earlier (Iwasa and Mohri, J. Biochem.94, 575–587, 1983).
• 3.3. The polypeptide spectrum of calmodulin target proteins changed significantly, e.g. the major polypeptides of 70 and 41 kDa increased, and the 200 and 43 kDa polypeptides decreased sharply during development from the two-cell to the early-gastrula stage.
• 4.4. According to our estimates, the molar concentrations of the calmodulin and targets were close enough, and therefore the Ca²⁺ signal should depend on the spatial-temporal distribution of free calmodulin in the cells.

     

Effect of diesel fuel hydrocarbons on embryogenesis and 45Ca2+ uptake by unfertilized eggs of sea urchin,Strongylocentrotus intermedius

1. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal concentrations (0.3–0.04 mg/l), was assessed through observation of embryogenesis and the intensity of ⁴⁵Ca²⁺ uptake.2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control.3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumu- lated 30–60% more ⁴⁵Ca²⁺ than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals.4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the ⁴⁵Ca²⁺ uptake by unfertilized eggs of control animals (30% more than in untreated eggs).5. Ionomycin-induced (concentration 10⁻⁸−10⁻⁹) increase of ⁴⁵Ca²⁺ uptake by unfertilized eggs (50–100% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure.6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.     

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.     

Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.     

Lipid composition of gametes and embryos of the sea urchin Strongylocentrotus intermedius at early stages of development

The lipid composition of sea urchin gametes and embryos was examined in detail by micro thin-layer chromatography (tlc) and gas-liquid chromatography (glc). Lipids of unfertilized eggs contain 53.7% triglycerides, 33.2% phospholipids, and 9.4% cholesterol, while spermatozoa lipids consist of 65.0% phospholipids, 15.5% cholesterol, and no triglycerides. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (DPG), and lysophosphatidylcholine (LPC) were identified among the phospholipids of both eggs and spermatozoa. The major part of egg and embryo PE was present as plasmalogen. After fertilization and the first cleavage, phospholipid content decreased from 33.2 to 29.4%, but the amount of phospholipids returned to the 33.2% level by the blastula stage and reached 39.7% by the pluteus stage. Lipid class composition showed no qualitative changes during development, but concentrations of PE, PS, LPC, and cholesterol increased, while those of PC, PI, and triglycerides decreased during the process. The principal fatty acids of neutral and polar lipid fractions are 14:0, 16:0, 18:1, 18:4, 20:1, 20:4, and 20:5. Their relative content underwent some changes during development.     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

Mechanism regulating Ca2+-dependent mechanosensory behaviour in sea urchin spermatozoa

Flagellar movement of the sea urchin sperm is regulated by intracellular Ca(2+). Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca(2+) control. To elucidate the mechanism of Ca(2+) dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca(2+), the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca(2+)-imaging with Fluo-4 showed that the intracellular Ca(2+) was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca(2+) influx was induced by Ca(2+) channels and the Ca(2+) efflux was induced by a flagellasialin-related Ca(2+)-efflux system, plasma membrane Ca(2+)-ATPases and the K(+)-dependent Na(+)/Ca(2+) exchanger. The results show that the Ca(2+)-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.     

Site of nuclear DNA replication in embryonic cells of the sea urchin Strongylocentrotus intermedius]

In the sea urchin embryonic cells, all newly synthesized nuclear DNA (n-DNA) pulse-labeled by 3H-thymidine was found within DNA-membrane complex (DNA-mc) isolated by centrifugation of lysates of nuclei after their treatment with Sarkosyl, Brij-35, or sodium dodecylsulfate through neutral sucrose (10--30%) gradients. This attachment has been shown not to be an artifact due to the unspecific effect of the detergents or the destabilization of the secondary structure of n-DNA because the association of the exogenous 14C-DNA with nuclear membrane and chromatin did not occur during the isolation of the DNA-mc. n-DNA was not replaced from DNA-mc when the latter was isolated in the excess of unlabeled denatured DNA. n-DNA associated with DNA-mc behaved as a precursor of chromosomal DNA. It is suggested that in sea urchin embryonic cells the synthesis of nuclear DNA is carried out by the replicative complex attached to the nuclear membrane.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.