Zymogen activation: a new system for homogeneous ligand-binding assay.

In this ligand-binding assay procedure, sensitivity is enhanced by successive generation of enzyme active sites via two zymogens from the blood-coagulation cascade: Factor X and prothrombin. A protease fraction from Russell's viper venom (RVV) acts upon Factor X, initiating a two-step cascade that culminates in generation of thrombin, the activity of which is monitored with a chromogenic substrate. In the model presented here, the analyte of interest, biotin, is covalently coupled to Factor X. In the presence of avidin, a biotin-binding protein, RVV cannot initiate cascade activity; however, the inhibition can be competitively overcome by addition of free biotin to the reaction mixture. In a complete system, the dose-response curve is linear from 20 to 100 nmol of biotin per liter. Such an assay offers improved sensitivity over many radioisotope-independent immunoassay methods, and may be applicable to a wide variety of analytes.

Escherichia coli acetate kinase mechanism studied by net initial rate, equilibrium, and independent isotopic exchange kinetics.

The equilibrium constant of the reaction catalyzed by acetate kinase (E.C. 2.7.2.1.) was determined: K = (MgADP) (acetylphosphate)/(MgATP)(acetate) = 6.7 +/- 1.3 X 10 (-4) (pH 7.4, 25 degrees). The respective free nucleotides uncomplexed to magnesium inhibit the net reaction in both directions: competitively with the respective magnesium nucleotide (MgATP or MgADP) and noncompetitively with the co-substrate (acetate or acetylphosphate). Excess free magnesium also inhibits the net reaction in both directions. The inhibition is not competitive with the phosphoryl donor (MgATP or acetylphosphate) but is competitive with respect to the phosphoryl acceptor (acetate or MgADP). A 50-fold increase in concentration of reactants at equilibrium in 0.1 M Tris/HCl, pH 7.4, at 25 degrees resulted in a rise to plateau levels of the acetate equilibrium acetylphosphate exchange rate (measured with [U-14C]acetate) and of the ATP equilibrium ADP exchange rate (measured with [U-14C]ADP and 10-fold higher than acetate equilibrium acetylphosphate), suggesting that there is no compulsory order of binding of magnesium nucleotide and co-substrate and that all chemical transformation steps cannot be much slower than the dissociation steps. The ATP equilibrium ADP exchange rate was independent of the presence or concentration of co-substrate, whereas the acetate equilibrium acetylphosphate exchange reaction occurred only in the presence of magnesium nucleotide, and the rate was directly related to the degree of saturation of enzyme with magnesium nucleotide. The independent ATP equilibrium ADP exchange, which presumably involves a phosphoenzyme intermediate, was progressively inhibited by increasingly elevated MgADP concentrations (when MgADP/MgATP greater than or equal 5), but increasing MgATP/MgADP was not inhibitory, suggesting that MgADP may bind to nonphosphorylated as well as phosphorylated enzyme, but that MgATP cannot bind to phosphorylated enzyme. While direct transfer of the phosphoryl group between nucleotide and co-substrate in a concerted mechanism has not been ruled out, an "activated ping pong" mechanism can also be proposed which is compatible with the isotopic exchange and initial net rate kinetic results. This mechanism includes a phosphoenzyme intermediate and requires enzyme-bount MgADP for phosphorylation of the enzyme by acetylphosphate.

The properties and large-scale production of L-asparaginase from citrobacter.

An intracellular L-asparaginase with antitumour activity was purified from a strain of Citrobacter. The optimum conditions for enzyme production by fermentation on scales up to 2700 l were investigated. Highest enzyme yield was obtained in corn-steep liquor medium (9-2%, W/V) at 37 degrees C. Oxygen limitation was not necessary for high enzyme yield. A total recovery of 4-3% from nucleic-acid-free extract and a 180-fold increase in specific activity were obtained after purificaiton. The specific activity of the purified preparation was 45 i.u./mg protein. The enzyme hydrolysed D-asparagine and L-glutamine at 7 and 5%, respectively, of its activity toward L-asparagine, but L-glutaminase activity could be demonstrated only at substrate concentrations above 5 mM. The Km values for L-asparagine and D-asparagine were 2-6 X 10(-5) and 1-4 X 10(-4) respectively. The anti-lymphoma activity of the enzyme was demonstrated with Gardner lymphosarcoma and was found only slightly less potent that Crasnitin, the most active asparaginase so far tested in this system.