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We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

The analytical active-enzyme-centrifugation method permits the determination of the hydrodynamic properties of enzymes while they are fully active on their substrates: the centrifugation can be performed at the very dilute concentrations used in kinetic studies (&aysmp; 1 µg/ml): furthermore a purified enzyme preparation is not necessary and in many cases a crude extract is sufficient. This method is beginning to be used in several laboratories even though few of its experimental aspects have been published. This paper reports the details for carrying out, an activeenzyme-centrifugation run, discusses the experimental conditions anti details with artifacts and ways, which are easy, to overcome them. [ABSTRACT FROM AUTHOR]

Further evidence is presented in this paper concerning the occurrence of three different molecular species of acetyleholinesterase in the electric eel's electric organ. These species, A, C, and D, differ in their sedimentation coefficients (8.5 S, 14.2 S, and 18.4 S, respectively). We show that the whole of the tissue acetyleholinesterase activity can be solubilized by repeated homogenisation, in the form of A, C, and D. Homogenisations under different conditions of ionic strength, or after prolonged autolysis yield closely similar results: the ratios of A, C, and D are remarkably constant. Moreover ammonium sulfate precipitation does not affect these species. This raises the question of their relationship and significance. All three species are recognized by antibodies obtained from rabbit immured against the purified enzyme. We have also studied the solubilization of acetylcholinesterase by trypsin in electric tissue slices. This is very effective and directly yields species B. This species, which is homogeneous in sedimentation, but might contain a variety of slightly different protein molecules, can also be obtained by partial tryptic digestion of A, C, and D, The sedimentation coefficient of species B is very close to that of purified acetylcholinesterase and we have previously suggested that the two kinds of molecules may be similar. Even though this is probably correct, we have been able to detect a significant difference (0.8 S unit) in the sedimentation coefficients. [ABSTRACT FROM AUTHOR]

Under standard assay conditions, the replicative DNA polymerase partially purified from rat liver non-histone chromosomal proteins exhibited a marked preference for native DNA over heat-denatured DNA as the template. However, work reported in the present paper demonstrates that this template preference could be reversed by changing the enzyme to template ratio in the assay system. This change in template preference was observed when either the concentration of DNA polymerase or of DNA was varied. Furthermore, it is shown that this alteration in template specificity is not the direct result of the action of an alkaline endonuclease present in the DNA polymerase preparation. [ABSTRACT FROM AUTHOR]