Identification of internal autoproteolytic cleavage sites within the prosegments of recombinant procathepsin B and procathepsin S. Contribution of a plausible unimolecular autoproteolytic event for the processing of zymogens belonging to the papain family.

The steps involved in the maturation of proenzymes belonging to the papain family of cysteine proteases have been difficult to characterize. Intermolecular processing at or near the pro/mature junction, due either to the catalytic activity of active enzyme or to exogeneous proteases, has been well documented for this family of proenzymes. In addition, kinetic studies are suggestive of a slow unimolecular mechanism of autoactivation which is independent of proenzyme concentration. However, inspection of the recently determined x-ray crystal structures does not support this evidence. This is due primarily to the extensive distances between the catalytic thiolate-imidazolium ion pair and the putative site of proteolysis near the pro/mature junction required to form mature protein. Furthermore, the prosegments for this family of precursors have been shown to bind through the substrate binding clefts in a direction opposite to that expected for natural substrates. We report, using cystatin C- and N-terminal sequencing, the identification of autoproteolytic intermediates of processing in vitro for purified recombinant procathepsin B and procathepsin S. Inspection of the x-ray crystal structures reported to date indicates that these reactions occur within a segment of the proregion which binds through the substrate binding clefts of the enzymes, thus suggesting that these reactions are occurring as unimolecular processes

Poinsettia Waltz

Title surrounded by poinsettia on each side with leaves extending around and under composer and publisher nameshttps://scholarsjunction.msstate.edu/cht-sheet-music/7931/thumbnail.jp

A comparison of incompressible limits for resistive plasmas

The constraint of incompressibility is often used to simplify the magnetohydrodynamic (MHD) description of linearized plasma dynamics because it does not affect the ideal MHD marginal stability point. In this paper two methods for introducing incompressibility are compared in a cylindrical plasma model: In the first method, the limit $\gamma \to \infty$ is taken, where $\gamma$ is the ratio of specific heats; in the second, an anisotropic mass tensor $\mathbf{\rho}$ is used, with the component parallel to the magnetic field taken to vanish, $\rho_{\parallel} \to 0$. Use of resistive MHD reveals the nature of these two limits because the Alfv\'en and slow magnetosonic continua of ideal MHD are converted to point spectra and moved into the complex plane. Both limits profoundly change the slow-magnetosonic spectrum, but only the second limit faithfully reproduces the resistive Alfv\'en spectrum and its wavemodes. In ideal MHD, the slow magnetosonic continuum degenerates to the Alfv\'en continuum in the first method, while it is moved to infinity by the second. The degeneracy in the first is broken by finite resistivity. For numerical and semi-analytical study of these models, we choose plasma equilibria which cast light on puzzling aspects of results found in earlier literature.Comment: 14 pages, 10 figure

Construct, Merge, Solve and Adapt: Application to the repetition-free longest common subsequence problem

In this paper we present the application of a recently proposed, general, algorithm for combinatorial optimization to the repetition-free longest common subsequence problem. The applied algorithm, which is labelled Construct, Merge, Solve & Adapt, generates sub-instances based on merging the solution components found in randomly constructed solutions. These sub-instances are subsequently solved by means of an exact solver. Moreover, the considered sub-instances are dynamically changing due to adding new solution components at each iteration, and removing existing solution components on the basis of indicators about their usefulness. The results of applying this algorithm to the repetition-free longest common subsequence problem show that the algorithm generally outperforms competing approaches from the literature. Moreover, they show that the algorithm is competitive with CPLEX for small and medium size problem instances, whereas it outperforms CPLEX for larger problem instances.Peer ReviewedPostprint (author's final draft

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy

AbstractNitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40 258 ±2 and 40 388 ±2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr, species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting

Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain.

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate). Thus, in this sense the stabilization of the thioimidate does not reflect a similarity in structure between the bound thioimidate and the transition state

Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo

Plan of Bruce Hill Acres II, Cumberland, Maine, 1982

Plan of Bruce Hill Acres II, Cumberland, Maine was created by C. R. Storer, Inc. in 1982.https://digitalmaine.com/cumberland_plans/1300/thumbnail.jp

Plans and Profiles of Island View Drive and Ebb Tide Drive, Cumberland, Maine, 1967

Plan of Island View Drive and Ebb Tide Drive, Cumberland, Maine was created by C. R. Storer, Inc. in 1967.https://digitalmaine.com/cumberland_plans/1362/thumbnail.jp

Plan of Maple View Terrace, Route 88 and Carriage Road, Cumberland, Maine, 1967

Plan of Maple View Terrace, Route 88 and Carriage Road, Cumberland, Maine was created by C. R. Storer, Inc. in 1967.https://digitalmaine.com/cumberland_plans/1377/thumbnail.jp