Search

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca(2+), Mg(2+)-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn(2+) blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.

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 Zn(2+) 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.

The effect of ionic strength of the reaction medium on the pH optimum, specificity, and mechanism of action of the acid DNase isolated from mature eggs of the sea urchin Strongylocentrotus intermedius was studied. Changes in ionic strength of the reaction medium caused a displacement of the pH optimum of the enzyme to acidity or alkalinity. The region and range of this displacement depended on the buffer used and on the substrate structure. For single-stranded, duplex, and supercoiled forms of DNA, the pH optimum displacements were 1.0, 1.4, and 2.0 pH units, respectively. The pH optimum displacement changed the mechanism of action of the enzyme. Under optimum pH conditions, the enzyme cleaved supercoiled DNA only by the double-hit mechanism, and fragments of duplex DNA resulted due to the coincidence of breaks in opposite chains. On pH displacement to acidity, the enzyme acted by the mixed mechanism (single- and double-hit). And the quantitative ratio of products of the enzymatic hydrolysis of supercoiled DNA was significantly changed depending on the pH displacement to acidity or to alkalinity. The findings are explained by the effect of salt-dependent electrostatic interactions during the formation of a nonspecific DNA-enzyme complex.

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.

Ca2+-Mg2+-dependent deoxyribonuclease (deoxyribonucleate-5'-oligonucleotidehydrolase E. C. 3.1.4.5). Molecular weight of the enzyme is found to be 40 000 daltons isoelectric point--4.4. The enzyme degraded DNA only in the presence of bivalent cations. It hydrolyses preferentially native DNA with pH optimum 7.0-7.2 in the presence of Mg2+ ions. Ca2+ ions shift the pH optimum to 8.0-8.5. Combined addition of Ca2+ and Mg2+ ions results in a sinergic effect and changes the enzyme specificity to the secondary DNA structure. The enzyme hydrolyses both native and denatured DNA by the endonucleolytic type to form oligonucleotides with 5' terminal phosphate the content of tetra-octanucleotides being 80-85%.

An ATP-dependent DNAse was isolated from the cells of the sea urchin Strongylocentrotus intermedius embryos by chromatography on DEAE-cellulose and gel chromatography on Sepharose 4B. The enzyme was found homogeneous during polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined by gel filtration through Sepharose 6B and was equal to approximately 450,000. The sedimentation coefficient as determined by ultracentrifugation was equal to approximately 15S. The pH optimum during native DNA hydrolysis lies within the pH range of 6,5--9,0 and that during hydrolysis of denaturated DNA--within the pH range of 9,0--9,5. The enzyme was activated by ATP and dATP at the optimal concentration of 10(-4) M. Other nucleoside triphosphates did not substitute for ATP in this reaction. The hydrolysis of denaturated DNA occurred via the exonuclease way with a formation of short (di-, tri,- tetra- and penta-) oligonucleotides. The enzyme hydrolyzed native DNA according to the endonuclease type with predominant formation of high molecular weight polynucleotides.

Ca2+, Mg2+-dependent DNAse has been isolated in a homogeneous state from the embryos of the sea urchin Strongylocentrotus intermedius. The enzyme hydrolyzes DNA only in the presence of bivalent metal ions and is inhibited by EDTA and EGTA. The most effective activators are Mn2+ and Co2+. In the presence of Mg2+ + EGTA, Ca2+, Ba+2 and Sr2+ the DNA is hydrolyzed in a lesser degree. Mg2+ + Ca2+ as well as Mg2+ + Sr2+ produce a synergic activating effect. The concentration of Ca2+ making up to one half of the maximal activity in the presence of 5 mM MgCl2 decreases with an increase in DNA concentration from 1.10(-4) M Ca2+ for 1,7 . 10(-5) M DNA-P up to 3,0 . 10(-5) M Ca2+ for 16,0. 10(-5) M DNA-P. The value of V for enzymatic hydrolysis of DNA does not depend on the concentration of Ca2+, while Km(app) for the enzyme increases from 2,0 . 10(-5) M DNA-P for 5 . 10(-4) M Ca2+ up to 25 . 10(-5) M DNA-P for 1 . 10(-5) M Ca2+. The Km(app) of the enzyme for Mg2+ + EGTA is 3.2 . 10(-5) M DNA-P, for Ca2+--0,77 . 10(-5) DNA-P.

Supercoiled DNAs of the SV40 virus and phi X174 phage have been studied under the effect of ATP-dependent DNase purified from sea urchin embryos. The studies showed a relaxing activity of the enzyme in relation to the supercoiled DNA. The supercoiled DNA treated with the enzyme in the absence of ATP was ultracentrifuged in a linear (5-20%) alkali sucrose gradient and the DNA preparations obtained by Davis formamide technique were examined by electron microscopy to demonstrate accumulation of double stranded DNA (RFIII). After addition of ATP to the incubation mixture only relaxed (RFII) and supercoiled (RFI) molecules of circular DNAs were observed among the products of enzyme hydrolysis of supercoiled DNAs.