Preparation and use of the poly-l-lysine-gold probe: a differential marker of glomerular anionic sites

Summary The conditions required for the production of a polylysine-coated gold (PL-G) complex, which shows optimal sensitivity for the demonstration of tissue anionic sites, expressed under different conditions of pH have been investigated. Problems encountered with this complex have been compared with those found with other methods of conjugation of polylysine to colloidal gold. The performance of a bovine serum albumin (BSA)-stabilized PL-G complex was examined against other PL-G conjugates, including complexes that are commercially available, for the detection of heterogeneous glomerular anionic site populations, expressed at pH 2.5 and pH 7.0.     

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

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.