Characterization of a novel murine T cell-activating factor

Purified resting peripheral lymph node T cells can be activated to produce interleukin 2 (IL 2) and to proliferate in the presence of Concanavalin A (Con A) and an apparently novel lymphokine that we call T cell activating factor (TAF). TAF is biochemically distinct from IL 1, IL 2, IL 3, and other colony stimulating factors, IL 4 (BSF-1) and interferons. Furthermore, of the recombinant and natural cytokines tested, only IL 2 and TAF are active in the TAF assay. In the presence of Con A, TAF stimulates an increase in the steady-state level of IL 2 mRNA in T cells, the secretion of active IL 2 into the culture medium, and the proliferation of the T cells. We propose that TAF is a previously undescribed molecule the function of which is to stimulate IL 2 production by T cells that have encountered antigen, and we propose that TAF has an important role in primary T cell immune responses.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction.     

Enumeration of denitrifying microbial populations in turf

Summary Denitrifer populations of a silt and silt loam soil under a Kentucky bluegrass turf were enumerated using the most probable number (MPN) procedure. The influence of soil texture, soil depth, soil moisture, and additions of nitrate fertilizer on denitrifier populations were determined. Saturated soil conditions increased denitrifier populations 87-fold in the silt soil and 121-fold in the silt loam soil. Denitrifier populations did not differ significantly between soil depths and additions of fertilizer nitrate did not influence populations.