Cysteine metabolism in cultured tobacco cells

Transported l-[(35)S]cysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight.The (35)S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal l-[(35)S]cysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the (35)S label present as compounds other than cysteine.The initial step in cysteine degradation yielded pyruvate, sulfide, and presumably NH(4) (+). Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K(m) to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

Evidence for structural changes in vertebrate thick filaments induced by calcium

Paired sedimentation studies of isolated, native thick filaments at pH 6.8, I = 0.12 and in the presence of 0.3 mm-free Mg²⁺ show that the sedimentation coefficient increases with Ca²⁺ concentration (pCa² midpoint = 5.5), leveling off at pCa 4.7. The addition of ATP or ADP (5 mm) has no effect on the hydrodynamic changes induced by Ca²⁺. At much higher free Mg²⁺ concentrations (5 mm), the midpoint of the transition is shifted to pCa = 5.3. Viscosity measurements of the filament system under comparable conditions reveal a decrease in the relative viscosity over the same range of Ca²⁺ concentration. Synthetic filaments prepared from purified myosin free of C-protein also show the same behavior. Native filaments from which myosin heads have been removed by treatment with papain do not show Ca²⁺ dependence. The dependence of the sedimentation coefficient of filament on protein concentration, as measured by differential sedimentation, is unaffected by Ca²⁺, indicating that the changes in hydrodynamic properties are probably not related to aggregation of the filaments. The Ca²⁺ effects are reversible and are not observed on replacing Ca²⁺ by Mg²⁺. Binding studies carried out at low ionic strength reveal two binding sites for Ca²⁺ (K_a = 1.7 × 10⁵m^?1) per mole myosin within the filament and evidence is presented showing that the DTNB light chain is the site of binding. The combined results are interpreted as indicating that thick filaments of vertebrate muscle undergo conformational changes at physiological levels of Ca²⁺ and provide evidence for a Ca²⁺-sensitive regulatory mechanism at the level of the thick filament.     

Effect of drift sampler exposure time and net mesh size on invertebrate drift density in the Njoro River,Kenya

Although invertebrate drift is an important ecological process in lotic ecosystems, very little is known about it in Kenyan rivers. The primary aim of this study was to investigate the effect of driftnet mesh size and exposure duration on drift density in 2017. Drift samples were dominated by Chironomidae, Baetidae, Simuliidae, Caenidae and Culicidae. The 100 µm mesh driftnet had the highest mean invertebrate density, followed by the 250 µm and 500 µm nets. Invertebrate drift densities decreased with increased exposure time. This study demonstrates that sampler mesh size and exposure time should be taken into account when characterising invertebrate drift in streams. Future studies should consider sampling different biotopes and during different seasons.