Purification and Characteristics of Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-Dependent and Acid DNases from Spermatozoa of the Sea Urchin Strongylocentrotus intermedius

V. V. Shastina, N. I. Menzorova, Yu. T. Sibirtsev^*, and V. A. Rasskazov

Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, pr. 100 let Vladivostoku 159, Vladivostok 690022, Russia; fax: 7 (4232) 314-050; E-mail: sibir@piboc.dvo.ru

^* To whom correspondence should be addressed.

Received May 18, 2002

---

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.

---

KEY WORDS: sea urchin, spermatozoa, Ca²⁺,Mg²⁺-dependent DNase, Ca²⁺,Mn²⁺-dependent DNase, acid DNase, DNase II, apoptosis

---