Active and inactive forms of the transition-state analog protease inhibitor leupeptin: explanation of the observed slow binding of leupeptin to cathepsin B and papain

Leupeptin and similar peptide argininal (arginine aldehyde) transition-state analog protease inhibitors exist in three covalent forms in aqueous solution, the leupeptin hydrate (IH), a cyclic carbinolamine form (IC) generated by the addition of the guanidino epsilon N to the aldehydic carbon, and the free aldehyde form (IA). 1H NMR in D2O show their equilibrium concentrations to be 42, 56, and 2% for IH, IC (R and S enantiomers), and IA. The rates of conversion of (formula; see text) were determined by 1H NMR in D2O by trapping IA with semicarbazide. Application of a deuterium isotope effect of 2.8 led to rate constants in H2O for kC of 0.092 min-1 and kD of 0.73 min-1. The equilibrium concentration of IA and rates for kC and kD are then used to explain the lag phase in the inhibition of cathepsin B and papain by leupeptin. Two circumstances are observed. (i) At micromolar concentrations of leupeptin and papain the binding of leupeptin is biphasic with rate constants identical to kD and kC. (ii) At more dilute nanomolar concentrations of total leupeptin and proteases, the observed lag phase for approach to steady-state inhibition (with rate constant k') is now explained by the low values of the koff rate constants (0.072 min-1 for cathepsin B and 0.024 min-1 for papain) together with the extremely low concentrations of the active inhibitor form IA, with k' = kon[IA] + koff. While kon[IA] is slow, the second-order rate constant kon is found to be quite fast, 1.2 x 10(7) M-1 s-1 for cathepsin B and 1.8 x 10(7) M-1 s-1 for papain. Thus, the binding of leupeptin to cathepsin B and papain may show a lag phase, but this is not due to slow binding.     

Implications of a consensus recognition site for phosphatidylcholine separate from the active site in cobra venom phospholipases A2.

A model structure of Naja naja kaouthia cobra venom phospholipase A2 has been constructed by utilizing molecular modeling techniques. Analysis of the model and available biochemical data reveal the presence in this enzyme of a putative recognition site for choline derivatives in loop 57-70 made up of residues Trp-61, Tyr-63, Phe-64, and Lys-65, together with Glu-55. The magnitude and shape of the electrostatic potential in this binding site are approximately 80% similar to that in the McPC603 antibody binding site specifically recognizing phosphocholine. Docking studies indicate that the recognition site we now describe and the phosphocholine head of an n-alkylphosphocholine molecule are complementary both sterically and electronically, mainly due to anion-cation and cation-pi interactions. Moreover, binding enthalpies of n-heptylphosphocholine to this site are found to parallel the catalytic rate of pancreatic, mutant pancreatic, and cobra venom phospholipase A2 enzymes acting on dihexanoylphosphatidylcholine micelles, suggesting that it behaves as an activator site. This proposal is in keeping with the "dual phospholipid" model put forward to account for the phenomenon of interfacial activation. This novel site is also shown to be able to discriminate choline derivatives from ethanolamine derivatives, in accord with experimental data. On the basis of the results obtained, two functions are assigned to this putative activator site: (i) desolvation of the lipid-enzyme interface, particularly the surroundings of tyrosine at position 69 (Tyr-63), and (ii) opening of the entrance to the active site by means of a conformational change of Tyr-63 whose chi 2 angle rotates nearly 60 degrees.     

Topological mapping of the active sites of cytochromes P4502B1 and P4502B2 by in situ rearrangement of aryl-iron complexes.

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (NA) and D (ND) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-aryl-protoporphyrin IX NA:ND regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (less than 2:greater than 98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the ND regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the NA regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

Increased Resistance Against Oxidative Stress Is Observed During A Short Period of Renal Reperfusion After A Temporal Ischaemia

Reperfusion of rat kidney submitted to temporal ischaemia induces a decrease in glutathione content. Lipid peroxidation is not detected in kidney homogenates but microsomes obtained after periods of reperfusion longer than 60 minutes show increased malondialdehyde values correlated with high oxygen consumption and superoxide free radical generation. Microsomes obtained from kidneys submitted to 15 or 60 minutes of reperfusion are resistant to NADPH-induced lipid peroxidation but after 120 minutes of reperfusion an increased lipid peroxidative response is observed. Although the mechanism of the protection found in microsomes against the induction of oxidative stress in the first 60 minutes of reperfusion is unknown, it is postulated that this subcellular fraction plays an important role in the oxidative stress observed after longer periods of reperfusion.     

Certain withanolides from Iochroma gesnerioides antagonize ecdysteroid action in a Drosophila melanogaster cell line

Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10^–8 M, the ED₅₀ values for 2,3-dihydro-3-methoxywithaferin A, 2,3-dihydro-3-methoxywithacnistine, 2,3-dihydro-3-methoxyiochromolide and 2,3-dihydro-3-hydroxywithacnistine are 3.5 × 10^-5 M, 1 × 10^–5 M, 5 × 10^–6 M and 2.5 × 10^–6 M, respectively.     

A spawning aggregation of Nassau grouperEpinephelus striatus (Pisces: Serranidae) in the Mexican Caribbean

Synopsis An annual spawning aggregation of Nassau grouper,Epinephelus striatus, observed off the southern coast of Quintana Roo, Mexico, on full moon days in December and January, was surveyed during the winters from 1991 to 1993. A fish aggregation had been appearing at one traditional site off Mahahual for more than 80 years, but currently it forms at undetermined sites far from the original site. Fish migration was northwards in the fore reef border along the 14 m isobath and isolated groups were observed. Through visual assessment fish aggregations were found to include 200 to 500 individuals. The fishing on Nassau grouper aggregations has been active since 1910 by using hook and line; however, recently fishermen used gill nets. It is necessary to continue the research on this aggregation and establish management guidelines to protect the species.     

Cell death,gliosis, and synaptic remodeling in the hippocampus of epileptic rats

Seizures set in motion complex molecular and morphological changes in vulnerable structures, such as the hippocampal complex. A number of these changes are responsible for neuronal death of CA3 and hilar cells, which involves necrotic and apoptotic mechanisms. In surviving dentate granule cells seizures induce an increased expression of tubulin subunits and microtubule-associated proteins, suggesting that an overproduction of tubulin polymers would lead to a remodeling of mossy fibers (the axons of granule cells). In fact, these fibers sprout in the dentate gyrus to innervate granule cell dendrites, creating recurrent excitatory circuits. In contrast, terminal mossy fibers do not sprout in the CA3 field. Navigation of mossy fiber's growth cones may be facilitated by astrocytes, which would exert differential effects by producing and excreting cell adhesion and substrate molecules. In the light of the results discussed here, we suggest that in adult brain activated-resident astrocytes (nonproliferating, tenascin-negative, neuronal cell-adhesion molecule-positive astrocytes) could contribute to the process of axonal outgrowth and synaptogenesis in the dentate gyrus, while proliferating astrocytes, tenascin-positive, could impede any axonal rearrangement in CA3. © 1995 John Wiley & Sons, Inc.     

Influence of Leaf Age, Soil Moisture, VPD and Time of Day on Leaf Conductance of Various Musa Genotypes in a Humid Forest-Moist Savanna Transition Site

Leaf age effects on the leaf conductance to water vapour diffusionof the adaxial and abaxial leaf surfaces were measured in themorning and in the afternoon on 17 different plantain and banana(Musa spp.) genotypes. The irradiance levels increased three-foldwhile leaf to air vapour pressure deficit levels increased two-to four-fold from morning to afternoon during the sampling periodin a field site located in the humid forest-moist savanna transitionzone of Nigeria. Conductance values were reduced in older, andsenescing leaves relative to the young and mature leaves bothin the morning and in the afternoon. Conductances were higherfor the abaxial leaf surfaces than the adaxial surface and higherin the afternoon than in the morning, with some genotypic differences.Lower values of leaf conductance to water vapour in the afternoonunder a short dry spell was sufficiently variable (P 0·05)among the test genotypes to indicate potential adaptation totransient dry conditions. Differential and relative leaf conductanceadjustments were noted among genotypes experiencing a shortdry spell versus non-limiting soil moisture conditions. Significantgenotypic differences were observed for leaf conductance amongthe 17 genotypes during the afternoon on the lower leaf surfaceof younger leaves. ABB cooking banana cultivars 'Fougamou' and'Bluggoe' might be potentially promising cultivars for transientdry conditions while AAB plantain 'Bobby Tannap' and one ofits hybrids TMPx 582-4 could be very sensitive to short dryspells according to this evaluation.Copyright 1994, 1999 AcademicPress Musa spp., Musa hybrids, adaxial leaf surface, abaxial leaf surface, stomatal response     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.