Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Ultra-short acting beta-blockers: a proposal for the treatment of the critically ill patient

Beta-blockade is of proven value in the therapy of acute myocardial infarction but, unfortunately, may produce cardiac failure by removal of needed sympathetic support. The long duration of action of available blockers (hours) makes reversal of failure a complicated problem and precludes rapid modification of therapy to match changing autonomic conditions. To improve the safety and efficacy of beta-blockade in this setting we have developed the concept of ultra-short beta-blockade and have identified a novel beta-blocker (ASL-8052) which possesses a duration of action less than 15 minutes. This compound is cardioselective and possesses efficacy in an animal model of acute myocardial infarction. It, therefore, appears to be suitable for rapid attainment of controlled levels of beta-blockade via intravenous infusion and rapid recovery from beta-blockade if required by the clinical situation. The compound should, therefore, be useful for safe therapy in critically ill cardiac patients.     

(+)-Arctigenin,a lignan from Wikstroemia indica

A new lignan, (+)-aretigenin has been isolated from the roots of Wikstroemia indica (Nan-Ling-Jao-Hua) and identified as 8(R) 8′(S)-4′-hydroxy-3, 4,3′-trimethoxylignan-olid (9, 9′) on the basis of spectral evidence as well as a direct comparison with its enantiomer, (?)-arctigenin.     

Increased root exudation of14C-compounds by sorghum seedlings inoculated with nitrogen-fixing bacteria

Summary Organic components leaked fromSorghum bicolor seedlings (‘root exudates’) were examined by recovering¹⁴C labelled compounds from root solutions of seedlings inoculated withAzospirillum brasilense, Azotobacter vinelandii orKlebsiella pneumoniae nif-. Up to 3.5% of the total¹⁴C recovered from shoots, roots, and nutrient solutions was found in the root solutions. Inoculation with Azospirillum and Azotobacter increased the amounts of¹⁴C and decreased the amounts of carbohydrates in the root solutions. When sucrose was added as a carbon source for the bacteria, the increase of¹⁴C in the solutions did not occur. Quantities of¹⁴C found in the root solutions were proportional to amounts of mineral nitrogen supplied to the plants. Bacterial growth also was proportional to nitrogen levels. When sorghum plants were grown in soil and labelled with¹⁴CO₂, about 15% of the total¹⁴C recovered within 48 hours exposure was found in soil leachates.     

Glycerol kinase as a substitute for dihydroxyacetone kinase in a mutant of Klebsiella pneumoniae.

With dihydroxyacetone as the sole source of carbon and energy, constitutively synthesized glycerol kinase of the glp system supported aerobic growth of Klebsiella pneumoniae mutants lacking the inducible dihydroxyacetone kinase of the dha system. Glycerol kinase had an apparent Km of 0.01 mM for its physiological substrate and 1 mM for its surrogate substrate. However, the growth rate on dihydroxyacetone of cells relying on glycerol kinase increased with the concentration of the carbon and energy source up to 50 mM, suggesting that permeation is rate limiting.