The site of substrate and fructose 2,6-bisphosphate binding to rabbit liver fructose-1,6-bisphosphatase

The binding site(s) in rabbit liver fructose-1,6-bisphosphatase for the active site binding ligand, fructose 6-phosphate, and the inhibitor, fructose 2,6-bisphosphate, have been investigated by using nuclear magnetic resonance spectroscopy. The distance from a nitroxide spin label to the bound ligands and the distance from the structural metal site to the bound ligands are about the same within experimental error. These data indicate that the two ligands probably bind at the active site in the rabbit liver enzyme.     

Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe

Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins. We have modified this technique for the isolation of plant calmodulin-binding proteins. [³⁵S]-methionine was used instead of the inorganic [³⁵S]-sulfate, or¹²⁵I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large number of filters. Here we describe in detail a procedure for the production and purification of [³⁵S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The [³⁵S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II that was previously isolated using a [¹²⁵I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive clone encoding a calcium-dependent calmodulin-binding protein was isolated.     

Nuclear magnetic resonance studies of fructose 2,6-bisphosphate and adenosine 5'-monophosphate interaction with bovine liver fructose-1,6-biphosphatase

1H and 31P nuclear magnetic resonance was used to investigate the interaction of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) with bovine liver fructose-1,6-bisphosphatase. Mn2+ bound to fructose-1,6-bisphosphatase was used as a paramagnetic probe to map the active and AMP allosteric sites of fructose-1,6-bisphosphatase. Distances between enzyme-bound Mn2+ and the phosphorus atoms at C-6 of fructose-6-P and alpha-methyl-D-fructofuranoside 1,6-bisphosphate were identical, and the enzyme-Mn to phosphorus distance determined for the C-6 phosphorus atom of Fru-2,6-P2 was very similar to these values. Likewise, the enzyme-Mn to phosphorus distances for Pi, the C-1 phosphorus atom of alpha-methyl-D-fructofuranoside 1,6-bisphosphate, and the C-2 phosphorus atom of Fru-2,6-P2 agreed within 0.5 A. The distance between enzyme-bound Mn2+ and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of Fru-2,6-P2 caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 s-1. It was concluded that Fru-2,6-P2 strongly affects the interaction of AMP with fructose-1,6-bisphosphatase and that the sugar most likely acts at the active site of the enzyme.     

The Biochemical Response of Electrical Signaling in the Reproductive System of Hibiscus Plants

Stimulation of the stigma of Hibiscus flowers by pollen, wounding (heat), or cold shock (4[deg]C) evokes electrical potential changes in the style, which propagate toward the ovary with a speed of 1.3 to 3.5 cm s-1. Potential changes were measured intracellularly by microelectrodes inserted in the style. The resting potential ranged from -90 to -112 mV (n = 20) in cells of the vascular tissue and from -184 to -220 mV (n = 22) in cells of the pollen-transmitting tissue. The amplitude of the potential changes was between 40 and 150 mV, depending on the kind of stimulus. Self- as well as cross-pollination hyperpolarized the resting potential after 50 to 100 s, followed by a series of 10 to 15 action potentials. In contrast, cooling of the stigma caused a single action potential with a different shape and duration, whereas wounding generated a strong depolarization of the membrane potential with an irregular form and a lower transmission rate. To determine the physiological function of the different signals measured in the style, the gas exchange and metabolite concentrations were measured in the ovary before and 10 min after stimulation of the stigma. Self- and cross-pollination caused a transient increase of the ovarian respiration rate by 12%, which was measured 3 to 5 min after the stigma was stimulated. Simultaneously, the levels of ATP, ADP, and starch increased significantly. In contrast, both cold shock and wounding of the stigma caused a spontaneous decrease of the CO2 content in the measuring chamber, as well as reduced metabolite concentrations in the ovary. Since the transport of labeled auxin from the top to the base of the style lasts at least 45 min, the influence of a chemical substance transmitted within 10 min is unlikely. Thus, our results strongly support the view that different, stimulus-dependent electrical signals cause specific responses of the ovarian metabolism.