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.     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

The hypothalamo-choroidal tract

Summary Light and electron microscopic examination of choroid plexuses from lateral ventricles of water-deprived and subcutaneously or intravenously vasopressin administered rats reveal morphologic changes typical for vasopressin responsive fluid transporting epithelia during hormonal stimulation. Ultrastructural changes noted in both dehydrated and vasopressin treated animals included: the frequent occurrence of choroidal dark cells, dilatation of the lateral and basal intercellular spaces, increased vacuolization of the apical cytoplasm, and a change in microvillar conformation from the normal clavate type to those with a filiform shape. On the basis of the ultrastructural changes observed it is proposed that the choroid plexus be regarded as a target tissue for vasopressin. These findings indicated that a vasopressinmediated transchoroidal cerebrospinal fluid absorption capability exists.Supported by U.S.P.H.S. Grant HD-08867This work submitted as partial requirement for the Master of Science degree in the Department of Anatomy, Colorado State University     

Distribution and developmental change in [3H]MK-801 binding within zebra finch song nuclei.

In many songbirds, vocal learning depends upon appropriate auditory experience during a sensitive period that coincides with the formation and reorganization of song-related neural pathways. Because some effects of early sensory experience on neural organization and early learning have been linked to activation of N-methyl-D-aspartate (NMDA) receptors, we measured binding to this receptor within the neural system controlling song behavior in zebra finches. Quantitative autoradiography was used to measure binding of the noncompetitive antagonist [3H]MK-801 (dizocilpine) in the brains of both adult and juvenile male zebra finches, focusing on four telencephalic regions implicated in song learning and production. Overall, the pattern of MK-801 binding in zebra finches was similar to the pattern found in rats (Monaghan and Cotman, 1985, J. Neurosci. 5:2909-2919; Sakurai, Cha, Penney, and Young, 1991, Neuroscience 40:533-543). That is, binding was highest in the telencephalon, intermediate in thalamic regions, and virtually absent from the brain stem and cerebellum. The telencephalic song areas exhibited intermediate levels of binding, and binding in the juveniles was not significantly different from adult levels in most song nuclei. However, in the lateral magnocellular nucleus of the anterior neostriatum (IMAN), binding at 30 days of age was significantly higher than binding in adults. Given the established role of NMDA receptors in other developing neural systems, both their presence in song control nuclei and their developmental regulation within a region implicated in song learning suggest that NMDA receptors play a role in mediating effects of auditory experience on the development of song behavior.     

Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

Relative sensitivity of three species of woody plants to SO2 at high or low exposure temperature

Summary Seedlings of paper birch (Betula papyrifera Marsh.), green ash (Fraxinus pennsylvanica Marsh.), and red pine (Pinus resinosa Ait.) fumigated with 0.2 ppm SO₂ for 30 h at 30° C had higher leaf diffusive conductances (LDC) and absorbed more sulfur than seedlings fumigated at 12° C. Comparisons among the three species fumigated at the same temperature, however, do not support the view that a plant with higher LDC should absorb more SO₂ than a plant with lower LDC. Mean relative growth rates ( ) of seedlings grown at 21° C after fumigation were variously affected by SO₂. of green ash was not inhibited by SO₂, but of roots of red pine seedlings was reduced by SO₂, with greater inhibition in seedlings fumigated at 30° C. Root and shoot of paper birch seedlings were lowered by SO₂, and effects of SO₂ were about equal at both exposure temperatures. The data indicate that temperature can affect mechanisms of SO₂ avoidance, tolerance, or both to various degrees in different species. Thus generalizations on the influence of exposure temperature on resistance of plants to SO₂ may be inappropriate.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison.