Changes In Proteinase Activities Research Articles

Methamidophos Influences Midgut Proteinase Activity and Subcellular Structures in the Wolf Spider Pardosa pseudoamulata (Araneae: Lycosidae).

A piezoelectric quartz crystal impedance (PQCI) sensor was used to investigate influences of the insecticide methamidophos on proteinase activity in midguts of the wolf spider, Pardosa pseudoamulata (Araneae: Lycosidae). Results from PQCI indicated that low-concentration dose methamidophos (0.008%) can activate the proteinase but high-concentration dose methamidophos (0.016-0.032%) can inhibit the enzyme activity. The changes in subcellular structure of spider midgut cells were also observed. Electron micrographs of spider midgut epithelial cells showed that the low-dose methamidophos did not visibly impact the structure of these cells. Conversely, high-concentration dose methamidophos led to severe changes in the cell structure, including the karyotheca dissolved, the nucleolus, and the endoplasmic reticulum disappeared. These may contribute to changes in proteinase activity of spider. This work documents a feasible method for rapid and reliable detection of proteinase activity.

The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor.

Scleral Matrix Metalloproteinases, Serine Proteinase Activity and Hydrational Capacity are Increased in Myopia Induced by Retinal Image Degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M rin equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M rzymographic activities at 50, 46 and 37 kDa M rare collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M rare increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (∼2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Identification of an interleukin-1 beta converting enzyme-like activity that increases upon treatment of P19 cells with retinoic acid as the proteasome.

We examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1 beta converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.

26S multicatalytic proteinase complexes decrease during the differentiation of murine erythroleukemia cells

Changes in multicatalytic proteinase activity during differentiation were investigated using Me 2SO-induced differentiation of murine erythroleukemia cells as a model. The apparent ATP-dependent multicatalytic proteinase activity decreased in the Me 2SO-treated cells with ATP-dependent incorporation of [ 3H]diisopropyl fluorophosphate decreasing notably after Me 2SO-treatment. This decrease in activity does not seem to arise from a cessation of cell-proliferation, because no significant changes in proteinase activity were observed under different culture conditions. Hydroxyapatite column chromatography was employed to analyze the form of multicatalytic proteinase. It was clearly demonstrated that the 26S form of the proteinase decrease in the differentiated cells relative to normal cells. Multicatalytic proteinase-associated proteins that bind to the proteinase in an ATP-dependent manner were purified on an anti-multicatalytic proteinase IgG conjugated column. Only a small amount of protein was recovered from the differentiated cells. These results suggest that the decrease in multicatalytic proteinase-associated proteins that occurs upon cell-differentiation abolishes the ATP-dependent activity of the proteinase.

Calcium-Dependent and Calcium-Independent Gelatinolytic Proteinase Activities of the Rat Ventral Prostate and its Secretion: Characterization and Effect of Castration and Testosterone Treatment1

Calcium-dependent and calcium-independent proteinase activities were detected in extracts of rat ventral prostate and its secretion by use of gelatin-containing SDS-PAGE zymography. Ca(2+)-independent proteinase activities of 22, 26, and 73-79 kDa and Ca(2+)-dependent activities of 58, 63, and 66 kDa were found in the adult gland. The 26- (most intense activity of gland) and 22-kDa activities were present in secretion and were not expressed in the undifferentiated gland of the 10-day-old animal. The Ca(2+)-dependent activities were also present in the secretion, where the 63-kDa form was more prominently expressed than the 58- and 66-kDa bands. The Ca(2+)-dependent and Ca(2+)-independent proteinase activities both responded to a broad range of pH values in the incubation media. The 73-79-kDa Ca(2+)-independent activities were sensitive to benzamidine and the Ca(2+)-dependent activities were inhibited by EDTA and EGTA. Both Ca(2+)-dependent and Ca(2+)-independent proteinase activities responded to androgenic manipulations. Castration was followed by the appearance of a 35-kDa Ca(2+)-independent proteinase (at 2 days) and a 43-kDa Ca(2+)-dependent proteinase (at 4 days). In the Ca(2+)-independent proteinase group, the 73-79-kDa activities were increased somewhat and the 22- and 26-kDa activities decreased after castration. The Ca(2+)-dependent proteinases of 58, 63, and 66 kDa increased in activity with castration, but activity of the 58-kDa form decreased again at 7 days after castration. Treatment of animals upon castration for 4 days with hydrocortisone prevented these changes in proteinase activities whereas treatment with actinomycin D or tranexamic acid did not. Testosterone propionate replacement therapy of rats castrated for 16 days stimulated the activities of the 22- and 26-kDa and 73-79-kDa Ca(2+)-independent and the 58- and 63-kDa Ca(2+)-dependent proteinases with 4 days of therapy. The activities of the 35-kDa Ca(2+)-independent and the 43-kDa Ca(2+)-dependent proteinases were repressed with 8 days of testosterone treatment. Thus, individual proteinases show differential changes in activity during development and in response to androgenic manipulation: this suggests that in addition to proteinases which are secreted, others may be involved in intracellular functions or in mediating tissue organization changes.

Activities of proteinases in maize and soybean roots in response to anoxic stress

This study characterized the changes in proteinase activities in maize inbred line H60 and soybean cultivar Keller roots in response to anoxia. After 24 h of anoxia, crude protein extracts from both maize and soybean root tips (10 cm) were assayed for proteinase activities at pHs ranging from 4.5 to 10.2. In anoxic roots of both maize and soybean, activities of proteinases with alkaline pH optima increased, and activities of proteinases with acidic pH optima declined. Proteinases with neutral pH increased in anoxic maize roots, but declined in anoxic soybean roots. Whether the differences in proteinase activities in anaerobic maize and soybean roots contribute to the differental susceptibility of the two species requires further study.

Changes in proteinase activities during the differentiation of murine erythroleukemia cells

Changes in intracellular proteinase activities were examined during DMSO-induced differentiation of murine erythroleukemia cells. Suc-APA-MCA hydrolytic activity was significantly decreased, and apparent ATP-dependent multicatalytic proteinase activity was also decreased with MEL cell differentiation. Cathepsin B and L activity was mainly present in the microsomal fraction of control cells, but a part of this activity had shifted to the lysosomal fraction of differentiated cells. With the translocation of cathepsin B from the microsomal to the lysosomal fraction, the pro-enzyme form of cathepsin B was converted into the mature enzyme. These results suggest that the lysosomal pathway contributes to the degradation of specific proteins with cell differentiation.

Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocindiabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in V max (i.e., maximal enzyme activity) without a change in K m (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.

Changes in proteinase activities and subcellular distribution during inactivation of alcohol oxidase in Candida boidinii.

Adaptation of methanol-grown Candida boidinii to ethanol utilization was accompanied by an increase in proteolytic activities, which behaved like known vacuolar enzymes. Degradation of alcohol oxidase protein was partially prevented by the serine proteinase inhibitor phenylmethanesulphonyl fluoride, but not by the carboxyl proteinase inhibitor pepstatin. Fractionation of cell-free extracts, by high-speed zonal centrifugation, of methanol-grown C. boidinii showed non-sedimentable and sedimentable proteolytic activities. Naturally occurring inhibitors of vacuolar proteinases were non-sedimentable. Fractionation of extracts prepared from methanol-grown cells which had been adapted to ethanol utilization for 5 h revealed significant changes in the sedimentability and distribution of proteolytic and acid phosphatase activities. These results suggest the possible involvement of a vacuolar process during alcohol oxidase degradation.

The relationship between proteolytic activity and loss of soluble protein in rice leaves from anthesis through senescence

The relationship between proteinase activity and loss of soluble protein from leaves was examined in the top four leaves on the main stems of rice from anthesis through senescence. During the period of rapid loss of soluble protein in the early and middle stages of senescence, changes in the proteinase activity were relatively small in these leaves: the activity slightly increased or fluctuated to some extent. However, the activity increased remarkably in all four leaves in the last stage of senescence although only a small amount of soluble protein had remained in these leaves. Rice plants were cultured with different amounts of nitrogen application and changes in proteinase activity in the third leaves from flag leaves were followed during senescence. The changes in the activity in the leaves were small during the period of rapid loss of soluble protein regardless of the status of nitrogen nutrition. These results indicate that proteinase activity is not necessarily correlated with the rate of protein l...

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization.

AbstractThe pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase).When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly.In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.