The Actions of Calmodulin Antagonists W-7 and TFP and of Calcium on the Gating Kinetics of the Calcium-activated Large Conductance Potassium Channel of the Chara Protoplasmic Drop: A Substate-sensitive Analysis

The effects of the calmodulin antagonists W-7 and trifluoperazine have been measured on the Ca²⁺-activated potassium channel in the membrane surrounding protoplasmic drops expressed from internodal cells of charophyte plants. The large-conductance (170 pS), voltage- and Ca²⁺-dependent gating, and prominent conductance substrate of this channel shows a strong kinetic resemblance to those of the Maxi-K channel from animal cells. This is the first study of the action of calmodulin antagonists which measures their effects on the most populated substates as well as the closed and main open states of Maxi-K channels. The substate analysis provides new evidence for different modes of action of- and different bindings sites for these calmodulin antagonists. Neither antagonist produces the simple closure of the channel reported previously as its effect on the Maxi-K channel, though both do induce flicker-block, reducing the mean current to near zero at high concentrations following an inverted Michaelis-Menten curve. W-7 reduces residence time in the fully open state, thus raising, in the same proportions, the probabilities of finding the channel in the closed state or a pre-existing substate. Its binding to the channel is voltage- and calcium-dependent. In contrast, trifluoperazine reduces residence in the open state and promotes an apparently new substate which overlaps the closed state at −50 mV but is distinguishable from it at voltages more negative than −100 mV. This substate may represent times that trifluoperazine is bound to the channel. Both antagonists have effects clearly distinguishable from that of withdrawing calcium from the channel, which does not affect open state residence time but increases closed state residence time. Thus neither antagonist reverses the activating effect of Ca²⁻. This is good kinetic evidence against the view that the channel is activated by Ca²⁺-calmodulin and that the effect of a calmodulin antagonist is to reverse this process by making Ca²⁻-calmodulin less available. Received: 26 August 1996/Revised: 7 October 1996     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .     

Further purification and characterization of human 3-methyladenine-DNA glycosylase Evidence for broad specificity

Further purification of a human placental 3-methyladenine-DNA glycosylase by phosphocellulose column chromatography yielded a 6000-fold increase in specific activity with greater than 5% recovery. Although 3-methyladenine was the predominant base released from double-stranded methylated DNA by this enzyme, minor releasing activities for 7-methylguanine and 3-methylguanine were also observed. During purification, the three DNA glycosylase activities consistently copurified with constant ratios of specific activity. Moreover, all the activities were heat-inactivated at 50°C at the same rate, required double-stranded methylated DNA as substrate, were inhibited by spermine and spermidine, and were not subject to product inhibition. These data strengthen the likelihood that the three activities are associated with a single DNA glycosylase.     

Effect of gibberellins and the growth retardant CCC on the nodulation of soya

Summary The effect of exogenous applications of gibberellins (GAs) or the growth retardant -chloroethyltrimethylammonium chloride (CCC) on root nodule formation and activity (C₂H₂-reduction) in soya was studied. Daily foliar application of GA₃ (2.89×10^–6 M) delayed the formation of nodule initials and reduced the numbers mass nodule^–1 and specific activity of nodules by 43%, 31% and 47% respectively, without affecting plant growth. Similar effects on nodulation were produced by foliar application of GA₄ (3.01×10^–5 M) or GA₇ (3.03×10^–5 M), or by the addition of GA₃ (2.89×10^–6 M) to the rooting medium. GA effectiveness in reducing nodule numbers was decreased by delaying its application until after the initial infection process had occurred, but the nodules formed were smaller and less active than those of the untreated control plants. The GA effect on nodulation and nodule activity was not associated with alterations in root exudate or due to a direct inhibitory effect of the hormone on the nitrogenase system. When the endogenous root content of GA-like substances was reduced (86% decrease) by foliar application of CCC (6.30×10^–5 M), nodule numbers were increased by 56%, but nodule size and total nodule activity were similar to those of control plants. The GA and CCC treatments had no effect on rhizobial growth in liquid culture nor on root colonisation by rhizobia.The results suggest that the endogenous content of root GA may have a regulatory role in both the infection process and in subsequent nodule morphogenesis, thus controlling both the number and effectiveness of the root nodules formed.     

Time-resolved microfluorescence for measuring coenzyme F420 in Methanobacterium thermoautotrophicum

Abstract The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F₄₂₀ and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F₄₂₀ fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F₄₂₀ from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

O-glycosylated C-glycosylflavones from Passiflora platyloba

Five C-glycosylflavonoids including two new compounds, isovitexin 7-rhamnosylglucoside and isomollupentin 7-rhamnosylglucoside, were obtained from the leaves of Passiflora platyloba. The known flavonoids were isovitexin, vitexin and isomollupentin (6-C-arabinosylapigenin). In addition, the coumarin esculetin was isolated.     

Effects of taurine and γ-aminobutyric acid on akinesia and analgesia induced by D-Ala2-Met-enkephalinamide in rats

Effects of taurine or γ-aminobutyric acid (GABA) on akinesia and analgesia induced by D-Ala²-Met-enkephalinamide were investigated in rats. Administration of taurine (dose range: 2.375×10^?2 M–9.5×10^?2 M/10 μl) into the left lateral ventricle 10 min prior to the injection of D-Ala²-Met-enkephalinamide (50 μg/10 μl) produced a dose-dependent reduction in the duration of akinesia and to some extent of analgesia, as estimated at 30 min and 60 min following the enkephalinamide injection; at the first estimation-time (10 min), taurine did not alter the duration of akinesia or that of analgesia. The median effective dose (ED₅₀) for akinesia determined at 60 min after D-Ala²-Met-enkephalinamide was 5 times greater and that for analgesia assessed at the same time was 1.7 times greater in taurine-treated rats than the respective doses in control animals. Administration of GABA under similar experimental conditions produced a dose-dependent reduction in the duration of analgesia from the initial estimation time (10 min) following the injection of D-Ala²-Met-enkephalinamide. The ED₅₀ for analgesia determined at 30 min after D-Ala²-Met-enkephalinamide was 3 times greater in GABA-treated rats than in control animals. Unlike the effects of taurine, GABA did not alter the duration of akinesia. Neither the duration of akinesia nor that of analgesia was modified by taurine or GABA alone in rats tested 9 min after the injection of each amino acid. These findings suggest that taurine may promote a recovery from both akinesia and analgesia, while GABA decreases only the analgesia induced by D-Ala²-Met-enkephalinamide.