Search

The importance of DNA synthesis during the early development of the sea urchin egg is discussed, as compared with the constancy of many other cellular constituents. The amount of enzymes which deaminate dCMP and MedCMP has been determined in different stages of development of the sea urchin embryo. A striking decrease in the two deaminases was observed, see Fig. 6. The following hypotheses have been discussed. (1) A cycle of conversion of the pyrimidine deoxynucleotides may exist in multiplying cells. In this way an appropriate assortment of pyrimidine deoxynucleotides is produced for DNA synthesis. (2) The deamination of dCMP to dUMP may have a pacemaker function for the DNA synthesis.

Matrices containing ligands which interact with catalytic or allosteric sites have been successfuly used for the purification of the allosteric enzyme dCMP-aminohydrolase. This enzyme is eluted specifically by a competitive inhibitor from a matrix coated with ligands which interact with the catalytic sites of the enzyme. The allosteric activator elutes the enzyme from a matrix coated with hydrazide-UTP which behaves as an allosteric inhibitor exploiting the allosteric conformational changes of dCMP-aminohydrolase.

A DNA endonuclease has been purified from eggs of Asterias forbesi by a simple four-step-purification procedure. The purified enzyme is at least 96% pure and is free of phosphatase, phosphodiesterase, and RNase. It has a pH optimum of 6.5 and does not require divalent cations. The enzyme produces 3'-phosphoryl and 5'-hydroxyl end groups. The products of exhaustive hydrolysis can be grouped in two fractions. The first fraction, 40%, contains a small amount of mononucleotides and di-, tri-, tetra-, penta-, and hexanucleo-tides. The second fraction, 60%, contains oligonucleotides larger than hexanucleotides.

The relation between sulphydryl groups and enzyme activity, and between sulphydryl groups and changes of conformation of the enzyme molecule caused by allosteric effectors has been studied in deoxycytidylate aminohydrolase with a homogeneous preparation of the spleen enzyme. Binding of 4 moles of p-chloromercuribenzoate per mole is sufficient to inactivate the enzyme. Three different time rates of inactivation by p-chloromercuribenzoate are obtained for the enzyme, for the enzyme · dTTP-Mg complex and for the enzyme · dCTP-Mg complex. The 4 thiol groups on the enzyme responsible for the inactivation by p-chloromercuribenzoate are not available to the action of 5,5′-dithio-bis(2-nitrobenzoic acid). Experiments of titration of the enzyme –SH groups by 5,5′-dithio-bis(2-nitrobenzoic acid) give additional support to the contention that at least three conformers are involved in the regulation of the activity of deoxycytidylate aminohydrolase. The data suggest that –SH groups have a very important role in catalysis and regulation of the enzyme.