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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 nucleotide specificity of membrane-bound Na+- and K+-activated ATPase from ox brain was compared with that of the Na+-dependent phosphorylation reaction of ATPase protein. 32P]Phospbate is incorporated into enzyme protein from terminally-labelled [32P]nucleotide triphosphates (ATP, ITP, GTP), a process dependent on the presence of Na+. Subsequent addition of K+ ions to the test system is followed by a rapid decrease in incorporated radioactivity. Enzyme-bound phosphate exchanges its label rapidly with adenine nucleotides and slowly with inosine and guanosine nucleotides. ATP, ITP, and GTP are substrates of Na+- and K+-activated ATPase, The ratio of the reaction rates is ATP: ITP: GTP = 27: 2: 1 . An almost identical activation pattern of nucleotide triphosphate hydrolysis by Na+ ions in the presence of saturating concentrations of K+ is found with all substrates (Km = 10 mM NaCl). On the other hand, the optimal stimulation by K+ at saturating concentrations of Na+ is variable hydrolysis of ATP is increased 10 times, that of ITP 4 times and that of GTP 1.2 times, ADP inhibits both ATP and ITP hydrolysis. Only high concentrations of IDP (50 mM) inhibit ATP hydrolysis, whereas low concentrations of IDP inhibit ITP hydrolysis. Identical nucleotide triphosphate specificities were found for Na+ and K+activated ATPase from ox brain and from human red blood cells. It is concluded that all nucleotide triphosphates investigated are hydrolyzed via the same acceptor group. The Na+-dependent phosphorylation reaction of ATPase protein is considered as part of the overall reaction of membrane-bound Na+- and K+-activated ATPase. [ABSTRACT FROM AUTHOR]

Values for binding of ATP and ADP to myosin B and ATP to myosin A were determined from mixtures of these nucleotides and proteins by use of rapid filtration to separate bound and unbound nucleotides and by use of an ATP-regenerating system (ereatine phosphate­creatine phosphokinase). Calculation of the binding curves of ATP to 105 g of myosin B by the mass law binding expression using two sets of binding sites, n1 and n2, and with binding constants, k1=40 × 103 and k2=1.5 × 103 1/mole, respectively, yielded a curve of good fit to the experimentally determined points when n1=0.4 and n2=1.2 moles per l05 g. Similar calculation of data from binding of ATP to myosin A fielded a curve of best fit when n1=0.5 and n2=3.0 per 105 g with k1=5,500 and k2=550 1/mole. At 1 mM ATP, myosin A bound 50% more than myosin B. ADP at 1 mM appeared to saturate myosin B about 97%. The amounts of ADP bound to myosin B fitted a curve with one value of n which equaled 0.36. Multiplication of 105 g by the factors required to convert the above n values to integers fields combining weights of 250,000 and 200,000 for myosin B and myosin A. [ABSTRACT FROM AUTHOR]