Trypsin-induced ATPase Activity in Potato Mitochondria

Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg²⁺, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg²⁺-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60 C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.     

Trypsin-induced phospholipase activity in human platelets.

Trypsin mediates a release of arachidonic acid with resultant increase in O2 consumption (a reflection of cyclo-oxygenase activity) by whole human platelets that is similar to thrombin's effect on these cells. The trypsin and thrombin effects can be differentiated in two ways: (1) at saturating concentrations the measured effects of trypsin greatly exceed those of thrombin; (2) EGTA [ethanedioxybis(ethylamine)-NNN'N'-tera-acetate] augments the effect of thrombin but not of trypsin. Thus trypsin and thrombin probably act at different loci in the pathway that induces phospholipase activity in human platelets.     

Phenol-promoted structural transformation of insulin in solution

Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2 Zn----4 Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of Zn2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni2 which is unable to replace Zn2 in 4 Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2 Zn----4 Zn transformation. 2 Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4 Zn conformation. Phenol, o- and p-cresol, m-nitrophenol, Nipagin M and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation.     

Trypsin-induced increase in intracellular cyclic AMP of lymphocytes.

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.     

Trypsin-induced proteome alteration during cell subculture in mammalian cells

Background

It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.

Methods

In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.

Results

36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.

Conclusions

In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.     

Trypsin-induced masking of tetrodotoxin receptor of the sodium channels in mollusc neurones

At the early stage of trypsin treatment of mollusc neurones tetrodotoxin cannot block the Na⁺ current. In the course of further exposure of neurones to trypsin, tetrodotoxin-sensitivity is restored completely, so its temporal loss results from shielding rather than destruction of the tetrodotoxin-binding site. Pronase and papain do not affect the tetrodotoxin action on the Na⁺ current.     

Trypsin-induced masking of tetrodotoxin receptor of the sodium channels in mollusc neurons

At the early stage of trypsin treatment of mollusc neurons tetrodotoxin cannot block the Na+ current. In the course of further exposure of neurones to trypsin, tetrodotoxin-sensitivity is restored completely, so its temporal loss results from shielding rather than destruction of the tetrodotoxin-binding site. Pronase and papain do not affect the tetrodotoxin action on the Na+ current.     

Trypsin-induced aggregation of bovine pulmonary artery endothelial cells cultured on microcarriers

We studied adherence between 'luminal' surfaces of pulmonary artery endothelial cells by standard aggregometry techniques, widely used for measuring aggregation of platelets and granulocytes. Using suspensions of bovine pulmonary artery endothelial cells cultured on microcarrier beads, in an aggregometer, we found that trypsin caused endothelial aggregation. The aggregation response occurred at trypsin concentrations as low as 0.001%. The degree of trypsin-induced aggregation indicated by the magnitude of the change in light transmission through the endothelial suspensions was related to the trypsin concentration, reaching a maximum level at trypsin concentrations of 0.01%. We conclude that trypsin, even in very low concentrations, causes adherence between 'luminal' surfaces of pulmonary endothelial cells probably because the enzyme destroys cell surface proteins which are necessary to prevent intercellular adherence. The method we describe may be useful for studying cell-cell interactions of endothelium.     

A transformation clustering algorithm and its application in polyribosomes structural profiling

Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. ‘molecular sociology’, is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.     

Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei.

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.     

Changes in the structural organization of the surface membrane in malignant cell transformation

Summary Malignant transformation of normal cells resulting in agglutinability by the carbohydrate-binding protein Concanavalin A can be explained by three types of changes in the structural organization of sites on the surface membrane. There can be an exposure of cryptic sites, a concentration of exposed sites by a decrease in cell size, and a rearrangement of exposed sites without a decrease in cell size resulting in a clustering of sites.     

The fragment transformation method to detect the protein structural motifs

To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the betabetaalpha-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the betabetaalpha-metal binding motif and the treble clef motif. The betabetaalpha-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions.     

Alpha-to-beta structural transformation of ovalbumin: heat and pH effects

Ovalbumin is an important member of the serpin superfamily without inhibitory activity. The heat- and pH-induced -to- structural transformations of ovalbumin were investigated by means of circular dichroism and binding of ANS and Congo red dyes. The native ovalbumin shows a mixture of -helix and -sheet, while both the heat and alkali treatments are able to transform the native protein into a predominance of -sheet secondary structure. The free energy changes during transitions to the unfolded state are 5.19 kcal/mol from the native state and 4.00 kcal/mol from the heat-treated one. The binding abilities of the heat-treated and the alkali-treated forms to ANS and Congo red suggest that the altered forms exhibit hydrophobic exposure and intermolecular interaction. The results substantiate that the altered protein forms bearing increased -sheet structures are prone to aggregation, which is implicated in the pathogenesis of some conformational diseases.     

Proteolytic cleavage and structural transformation: Their relationship in bacteriophage T4 capsid maturation

Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp 23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp 23 monomers excised by phage-specific proteolysis. Cleaved 17^? giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.     

Trypsin-induced cell surface changes in ascidian embryonic cells: regulation of differentiation of a tissue-specific protein.

Developing animal quartets removed surgically from 8-cell stage of Ascidia malaca and Phallusia mamillata have been treated for a short time with a low concentration of trypsin. The result is a differentiation of some neural structures, of pigment spots and of a tissue-specific enzyme, brain pigment cell tyrosinase. Tyrosinase activity, as detected histochemically, appeared in the pigment cells some hours before the normal time independently of any inductive interactions with related embryonic tissues. A study with the electron microscope has given evidence of the presence of presumed nervous cells and melanin granules related to them. An autoradiographic study using [³H]uridine has demonstrated presumed RNA synthesis which suggests gene activation. The results are discussed in relation to the possible role of the plasma membrane during embryonic development.     

Lysozyme fibrillation: deep UV Raman spectroscopic characterization of protein structural transformation

Deep ultraviolet resonance Raman spectroscopy was demonstrated to be a powerful tool for structural characterization of protein at all stages of fibril formation. The evolution of the protein secondary structure as well as the local environment of phenylalanine, a natural deep ultraviolet Raman marker, was documented for the fibrillation of lysozyme. Concentration-independent irreversible helix melting was quantitatively characterized as the first step of the fibrillation. The native lysozyme composed initially of 32% helix transforms monoexponentially to an unfolded intermediate with 6% helix with a characteristic time of 29 h. The local environment of phenylalanine residues changes concomitantly with the secondary structure transformation. The phenylalanine residues in lysozyme fibrils are accessible to solvent in contrast to those in the native protein.     

Ultraweak thermalized neutron field as an underestimated factor of DNA structural transformation

It was shown that thermalized neutrons in superlow doses were capable of inducing detectable structural transformations in DNA films and DNA solutions.     

Trypsin-induced increase in cyclic AMP concentration in rat thymocytes. An effect independent of calcium and calmodulin.

Trypsin produces a dose-related increase in cellular cyclic AMP concentration in rat thymocytes [Shneyour, Patt & Trainin (1976) J. Immunol. 117, 2143-2149; Segal & Ingbar (1983) Clin. Res. 31, 277A]. In the present study, I examined whether this effect of trypsin requires Ca2+ and whether it is modified by calmodulin. In fresh thymocytes suspended in standard medium (containing 1 mM-Ca2+), trypsin produced a concentration-dependent increase in cytoplasmic free Ca2+ concentration, which was evident at a concentration of 50 micrograms of trypsin/ml and reached maximal values at about 1 mg/ml. This effect of trypsin was very prompt in onset, almost immediate, and reached maximal values within 2-3 min. But in cells suspended in essentially Ca2+-free medium (6 nM free Ca2+), trypsin had no effect on cytoplasmic free Ca2+ concentration, which indicates that trypsin acted by increasing Ca2+ uptake rather than Ca2+ release from an intracellular pool. However, the increase in thymocyte cyclic AMP concentration produced by trypsin was independent of extracellular Ca2+ and was not influenced by calmodulin, because it was the same in the presence or absence of Ca2+ and was not changed by the calmodulin inhibitor trifluoperazine. I therefore suggest that in rat thymocytes the trypsin-induced increase in cyclic AMP concentration does not require Ca2+ and is not influenced by calmodulin.     

Trypsin-induced alterations of insulin binding, microfilament organization and cell shape in fibroblastic cultures from non-diabetic and diabetic mice

Although fibroblastic cultures from the skin of both non-diabetic and diabetic (db/db) mice have specific receptors for insulin, cells from diabetic mice bind only half as much insulin as those from non-diabetic animals. Treatment of cultures from non-diabetic and diabetic mice with trypsin caused an increase in the total number of binding sites from 7.7 × 10⁴ to 11.0 × 10⁴ per cell in nondiabetic and from 2.9 × 10⁴ to 5.1 × 10⁴ per cell in diabetic cells. The increase is reversible and apparently specific for trypsin. Cells cultured from non-diabetic and diabetic animals are flat and fusiform and have microfilaments of various sizes occurring individually in the cytoplasm and in bundles running parallel with the plasma membrane. Trypsin treatment causes rounding of cells, with development of numerous blebs and folds, and depolymerization and disappearance of microfilament bundles in both non-diabetic and diabetic cells. The present study demonstrates that although diabetic fibroblasts have fewer insulin receptors than cells from non-diabetic littermates, the effects of trypsin on insulin receptors, organization of macrofilament bundles, and cell morphology have not been altered by the expression of the diabetic state.     

Accurate prediction of protein structural class using auto covariance transformation of PSI-BLAST profiles

Computational prediction of protein structural class based solely on sequence data remains a challenging problem in protein science. Existing methods differ in the protein sequence representation models and prediction engines adopted. In this study, a powerful feature extraction method, which combines position-specific score matrix (PSSM) with auto covariance (AC) transformation, is introduced. Thus, a sample protein is represented by a series of discrete components, which could partially incorporate the long-range sequence order information and evolutionary information reflected from the PSI-BLAST profile. To verify the performance of our method, jackknife cross-validation tests are performed on four widely used benchmark datasets. Comparison of our results with existing methods shows that our method provides the state-of-the-art performance for structural class prediction. A Web server that implements the proposed method is freely available at http://202.194.133.5/xinxi/AAC_PSSM_AC/index.htm.