1 The insecticidal activity of the neotropical pepper Piper tuberculatum Jacq. and its isolated piperamides was studied. Bioassays with the mosquito Aedes atropalpus L. assessed the relative toxicity of the whole extract of Piper tuberculatum and four of the piperamides, which were isolated and identified, then prepared synthetically. 2 The results confirm that P. tuberculatum leaf extracts are as effective as black pepper seed extract and provide an alternative pepper insecticide from a more convenient source, the leaves. 3 Experiments with piperamides showed that the tertiary and quaternary mixtures have greater-than-additive toxicity compared to single compounds or binary mixtures. One of the four amide compounds, 4,5-dihydropiperlonguminine, was the most acutely toxic in mosquito larvae bioassays. 4 A study of piperamide levels from different P. tuberculatum populations in Costa Rica determined that they were relatively homogeneous. Piper tuberculatum from only one of the five sites had higher levels of one piperamide, 4,5-dihydropiperine, in both leaf and stem parts. One explanation for differences in the amide concentrations between populations is that one site is ecologically unique compared to the other four.

The inhibition of the energy-linked nicotinamidenucleotide transhydrogenase (TH; EC 1.6.1.1) by dicyclohexylcarbodiimide (DCCD) has been further studied because of its important mechanistic implications. We had shown earlier that TH bound to submitochondrial particles from bovine heart is inhibited by DCCD and that NAD(H) protects the enzyme against this inhibition [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221]. By contrast, Pennington and Fisher [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] working with purified TH concluded that NAD(H) does not protect against DCCD inhibition and that DCCD inhibition involves the TH proton channel rather than the nucleotide-binding active site. The present study shows that NAD(H) as well as AMP and ADP, which are known to bind to the NAD(H) binding site from competitive inhibition studies, protect the purified TH against inhibition by DCCD, whereas 2'-AMP and 3'-AMP, which bind to the NADP(H) site on TH, do not protect. In addition, it is shown that whereas the unmodified TH binds to NAD-agarose such that it is elutable by buffer containing NADH, the DCCD-modified enzyme does not bind to NAD-agarose. These results suggest strongly that DCCD binds at or near the NAD(H) binding site on TH. Another less likely possibility is that NAD(H) and DCCD bind to separate sites, but their bindings are mutually exclusive. With the use of [14C]DCCD, it has been shown that 100% activity inhibition corresponds to 0.5 mol of DCCD binding per mol of TH (Mr approximately 11 X 10(4].(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of uncouplers and valinomycin plus nigericin (in the presence of K+) were studied on the apparent Km for substrates and apparent Vm. of the following energy-linked reactions catalyzed by submitochondrial particles: oxidative phosphorylation, NTP-33Pi exchange, ATP-driven electron transfer from succinate to NAD, and respiration-driven transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate. In all cases, partially uncoupling (up to 90%) concentrations ofuncouplers or valinomycin plus nigericin were found to decrease apparent Vm. and to increase apparent Km. Results plotted as In (Vme/Km) versus the concentration of uncouplers or ionophores showed a linear decrease of the former as a function of increasing perturbant concentration (i.e., decreasing free energy). Because Vm./Km may be considered as a measure of the apparent firstorder rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex, the results are consistent with the interpretation that membrane energization leads to a change in enzyme conformation with the resultant increase in enzyme-substrate affinity and facilitation of the reaction rate under consideration. The significance of these findings with respect to the mechanism of action of the energytransducing systems studied is discussed.Our previous studies ofenergy-linked transhydrogenation from NADH to two NADP analogs as catalyzed by submitochondrial particles (SMP) showed that membrane energization resulted in a 40-fold increase in Vm.jKm (decrease in apparent Km and increase in apparent Vm,) for both the reduced and oxidized substrates (1). The VmJ/Km increases were pH dependent, and the highest values were obtained at pH 7.5, the optimal pH for mitochondrial energy-linked processes. These results indicated that, in NADH -* NADP transhydrogenation, enzyme-substrate affinity as reflected by Vmj,/Km (2) is modulated by the energized state of SMP and suggested that the basis of this energylinked modulation might be a A~uH+-induced enzyme conformation change resulting in an increased rate ofenzyme-substrate interaction and a consequent facilitation ofhydride ion transfer from NADH to NADP (1). In other studies, thermodynamic results consistent with the possible AuH'-induced conformation change of the transhydrogenase enzyme were also presented (3).In the present paper, we report on the modulation of Vm.and Km by uncouplers and by valinomycin plus nigericin for four energy-linked reactions of mitochondria. The reactions studied were (i) ATP synthesis, (ii) NTP-3Pj exchange, (iii) nicotinamide nucleotide transhydrogenation, and (iv) ATP-driven electron transfer from succinate (succinate/fuimarate Em,7 = +30 mV) to NAD (NADH/NAD Em,7 =-315 mV).METHODS AND MATERIALS SMP was prepared from bovine heart mitochondria essentially according to L6w and Vallin (4) as described elsewhere (1). Protein concentration was determined by the biuret method (5) in the presence of 1% potassium deoxycholate. The assay...

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