[Ribonuclease from the hepatopancreas of the red king crab Paralithodes camtschatica].

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg2+ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT50 = 53-55 degrees C) and were strongly inhibited by NaCl (IC50, 0.1-0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.

[Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas].

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphmonoesterases, one alkaline phosphodiesterase (AlkPD), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg2+ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80: AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID50 from 47 to 62 degrees C). AlkP is inhibited by NaCl (IC50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.

[Trofosides A and B and other cytostatic steroid-derived compounds from the Far East starfish Trofodiscus über].

Three new polar steroids identified as trofoside A, (20R,24S)-24-O-(3-O-methyl-beta-D-xylopyranosyl)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, its 22(23)-dehydro derivative (trofoside B), and 15-sulfoxy-(20R,24S)-5alpha-cholestane-3beta,6beta,8,15alpha,24-pentaol sodium salt, were isolated from Trofodiscus uber starfish extracts collected in the Sea of Okhotsk. Two known compounds, trofoside A aglycone, (20R,24S)-3beta,6alpha,8,15beta,24-pentahydroxy-5alpha-cholestane, and triseramide, (20R,24R,25S,22E)-24-methyl-3beta,6alpha,8,15beta-tetrahydroxy-5alpha-cholest-22-en-27-oic acid (2-sulfoethyl)amide sodium salt, were also found. The structures of the isolated polyoxysteroids were established from their spectra. Minimal concentrations causing degradation of unfertilized egg-cells of the sea-urchin Strongylocentrotus intermedius (C(min)) and terminating the cell division at the stage of the first division (C(min) embr.), as well as the concentrations causing 50% immobilization of sperm cells (ImC50) and inhibiting their ability to fertilize egg-cells by 50% (IC50) were determined for the isolated compounds. Of three compounds highly toxic in embryos and sea-urchin sperm cells, the polyol with a sulfo group in the steroid core was the most active; two glycosides with monosaccharide chains located at C3 and C24 atoms were less toxic. Note that all the compounds with the spermiotoxic activities differently affected the embryo development. The positions of monosaccharide residues in the core considerably influence the compound activity. For example, both mono- and double chained glycosides with the monosaccharide fragment at C3 and C24 atoms are active against sea-urchin sperm cells and embryos, whereas the C24 glycosylated trofoside A does not affect embryos and displays a poor spermiotoxicity.

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 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.

[Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos]. / Issledovanie substratnoi spetsifichnosti Ca2+,Mg2+-zavisimoi DNKazy iz émbrionov morskogo ezha Strongylocentrotus intermedius.

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 bivalent metal ions on enzymatic activity of Ca2+, Mg2+-dependent DNAse from sea urchin Stronglyocentrotus intermedius embryos]. / Vliianie ionov dvukhvalentnykh metallov na fermentativnuiu aktivnost' Ca2+,Mg2+-zavisimoi DNKazy iz embrionov morskogo ezha Strongylocentrotus intermedius.

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.

[Isolation and some properties of Ca2+-, Mg2+-dependent deoxyribonuclease from sea urchin (Strongylocentrotus intermedius) embryos]. / Vydelenie i nekotorye svoistva Ca2+, Mg2+-zavisimoi dezoksiribonykleazy iz embrionov morskogo ezha Strongylocentrotus intermedius

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%.