Protease Activity In Extracts Research Articles

Protease activities of extracts from kiwi of various colors and their use as a tenderizer in cured pork loins

The aim of this study was to evaluate protease activities of kiwi extracts with various colors (red, green, gold) and determine physicochemical and textural properties of pork loins added with such kiwi extracts during storage at 8°C for 24 h. Green and gold kiwi extracts exhibited higher protease activities than red kiwi extract using casein as a substrate. Pork loin was injected with 5% or 10% of red, green, or gold kiwi extract. Shear force were was decreased when kiwi extracts were added. When 10% of green or gold kiwi extract was added to pork loin, the tenderness increased with increased incubation time, which was confirmed by myosin heavy chain (MHC) band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). These results indicate that green and gold kiwi extract could be used as a tenderizer for though meat through proteolytic activities of enzymes contained in kiwi. Novelty impact statement Green and gold kiwi extracts exhibited higher protease activities than red kiwi extract. When 10% of green and gold kiwi extracts were added to pork loin, the tenderness increased with incubation time. This study demonstrates that green and gold kiwi extracts could be used as a meat tenderizer without affecting the physicochemical properties of meat products.

Nematotoxicity of drupacine and a Cephalotaxus alkaloid preparation against the plant‐parasitic nematodes Meloidogyne incognita and Bursaphelenchus xylophilus

Species of Cephalotaxus (the plum yews) produce nematotoxic compounds of unknown identity. Consequently, bioassay-guided fractionation was employed to identify the compound(s) in Cephalotaxus fortunei twigs and leaves with activity against plant-parasitic nematodes. A crude alkaloid extract, particularly drupacine, was responsible for much of the nematotoxicity. The ED50 of drupacine for Bursaphelenchus xylophilus was 27.1 µg mL⁻¹, and for Meloidogyne incognita it was 76.3 µg mL⁻¹. Immersion of M. incognita eggs in 1.0 mg mL⁻¹ crude alkaloid extract (the highest tested concentration) reduced hatch by 36%; immersion of second-stage juveniles (J2) resulted in 72-98% immobility. Crude alkaloid extract and drupacine suppressed protease activity in extracts of the microbivorous nematode Panagrellus redivivus by 50% and 80%, respectively. Application of 0.02-0.5 mg mL⁻¹ crude alkaloid extract to soil with M. incognita inoculum did not significantly reduce pepper plant shoot length or weight, compared with nematode-inoculated, water-treated controls, but the number of eggs and J2 per root system respectively decreased by 69% and 73% at 0.5 mg mL⁻¹. Drupacine and a crude alkaloid extract suppress nematode hatch, activity of mixed life stages, and population numbers on plant roots. This is the first demonstration of nematotoxicity of crude Cephalotaxus alkaloids and drupacine.

Dual origin of gut proteases in Formosan subterranean termites ( Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite ( Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.

A novel Ca2+-activated protease from germinating Vigna radiata seeds and its role in storage protein mobilization

Calcium (Ca(2+))-dependent/activated proteases make decisive cleavages in proteins, affecting their further degradation/activation. Few such Ca(2+)-dependent proteases have been reported from plants, and none during germination-related events. Seeds are woken up from their quiescent state upon imbibition of water. The subsequent process of germination is strongly influenced by hormones (mainly gibberellins) and light, with both resulting in change in intracellular Ca(2+). We have investigated the effect of Ca(2+) on protease activity in extracts prepared from dry Vigna radiata (L.) Wilczec seeds and cotyledons 4, 24, 48 and 72h post-imbibition. Ca(2+)-activated protease activity is present at a very low level in dry seeds, rises with imbibition and peaks 24h post-imbibition. Subsequently, the activity rapidly declines, even as total protease activity continues to rise. Calcium activation of proteolysis was reversed by ethylene diamine tetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 1,10, phenanthroline, chlorpromazine and by beta-mercaptoethanol in a concentration-dependent manner. Protease activity was also inhibited by para chloro mercuribenzoate (pCMB) and l-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E 64), while phenyl methyl sulfonyl fluoride (PMSF) and pepstatin did not effect Ca(2+) activation. The protease could be separated from the calmodulin fraction by size-exclusion chromatography, while retaining its ability for Ca(2+) activation, excluding the possibility of activation through calmodulin-based pathways. The presence of a Ca(2+)-activated protease in the cotyledons suggests its role in a predetermined program of germination involving elevation of cytosolic Ca(2+) levels during germination. This protease could be an important enzyme interfacing cytoplasmic signaling events and initiation of storage protein mobilization during seed germination.

Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini

Protease activities in extracts of Opisthorchis viverrini were investigated using gelatin zymography and fluorogenic peptide substrates. Using gelatin-impregnated X-ray film, 2 microg of O. viverrini excretory-secretory products (Ov-ES) and adult somatic extract (Ov-SE) showed proteolytic activity. Zymography of both O. viverrini extracts revealed bands at approximately 30 kDa. Using fluorogenic peptide substrates, the majority of O. viverrini activity was determined to be cathepsin L-like cysteine protease (cleaved Z-Phe-Arg-aminomethylcoumarin (AMC)) whereas little or no activity was ascribable to other classes of proteases. The O. viverrini cysteine protease activity was greatest at pH 6.0 and the activity was inhibited by the class-specific inhibitors, E-64 and Z-Ala-CHN2. Chromatographic purification of O. viverrini cysteine proteases on thiol-sepharose enriched for protein(s) of approximately 30 kDa from Ov-ES and Ov-SE. The activity profile of the purified enzyme was similar to that of the cathepsin L-like activity characterized in Ov-SE and Ov-ES. Furthermore, determination of cysteine protease activity in several developmental stages of the parasite revealed the highest protease activity in metacercariae soluble extract, followed by Ov-ES, egg soluble extract, and Ov-SE. These findings demonstrated that O. viverrini has a cathepsin L-like cysteine protease(s) and suggested that abundant cysteine protease activity was present in metacercariae where the hydrolase might be involved in cyst excystation during mammalian infection.

Occurrence of Digestive Cysteine Proteases in Perillus bioculatus, a Natural Predator of the Colorado Potato Beetle

Oryzacystatins (OCs) are protease inhibitors (PIs) that inhibit Colorado potato beetle (CPB) digestive proteases, and transgenic potato plants containing these PIs are currently under test. However, OCs could interfere with the digestive system of beneficial insects. Protease activity and susceptibility to class-specific protease inhibitors were studied in protein extracts of Perillus bioculatus, a stinkbug predator that has shown potential for biological control of the CPB. At physiological pH, the analysis of protease activity showed that up to 90% of P. bioculatus protease activity is of the cysteine type. All active life stages of the predator were tested, and electrophoretic characterization detected no major qualitative variation in protease pattern between stages. Protease activity in extracts of P. bioculatus nymphs was significantly reduced, up to 70%, by the two recombinant cystatins from rice (OCI and OCII), and by stefin A, a PI encoded by a human gene. These results clearly indicate that cysteine PIs are active not only against the CPB digestive protease complex, but also against proteases of one of its most important natural predators.

Bcl-2 Expression in Neural Cells Blocks Activation of ICE/CED-3 Family Proteases during Apoptosis

The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2. Rapid activation of a 17 kDa subunit of an activated member of the ICE/CED-3 family is demonstrated by affinity-labeling GT1-7 extracts from apoptotic controls cells with a biotinylated ICE/CED-3 inhibitor. This activation corresponds to an increased ICE/CED-3-like protease activity in extracts measured by a fluorogenic substrate assay. In a cell-free system, these extracts induce apoptotic morphological changes in intact nuclei. All three activities are readily inhibited by treatment of control extracts with ICE/CED-3-like protease inhibitors. Overexpressed Bcl-2 inhibits the activation of the 17 kDa protein, the ICE/CED-3-like protease activity in the fluorogenic assay, and the induction of apoptotic morphological changes in HeLa nuclei in the cell-free system, similar to results obtained with ICE/CED-3 protease inhibitors. At the mRNA level, overexpression of Bcl-2 did not alter expression of five members of the ICE/CED-3 family: CPP32, ICE, Mch 2, Nedd 2, and TX. Overexpression of Bcl-2 prevented the apoptosis-induced processing of pro-Nedd 2 to the cleaved form. These data suggest that Bcl-2 participates upstream from the function of ICE/CED-3 proteases and may inhibit apoptosis by preventing the post-translational activation of ICE/CED-3 proteases.

A Comparison of Trypsin‐like Protease Activities in Extracts of Human and Murine Cytolytic T Cells

A Comparison of Trypsin‐like Protease Activities in Extracts of Human and Murine Cytolytic T Cells

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice.

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.

Substrate Specificity of Enzymes from Papaya Callus Cultures

Summary Optimal protease activity in extracts of callus cultures of papaya is obtained by triturating lyophilized callus powder with phosphate buffer at pH 6.0 in a glass mortar; addition of reducing agents to the extraction medium does not increase or stabilize enzyme activity. Casein, casein yellow, azocasein and azoalbumin are good substrates for the proteases; little or no acitivity could be measured with hemoglobin, azocoll, hide powder azure and benzoyl-DL-arginine-p-nitroanilide (BAPA) as substrate.

Neutral protease from the polymorphonuclear leucocytes of human rheumatoid synovial fluid.

1. The cartilage-proteoglycan-degrading activity of synovial fluid cells from rheumatoid patients is primarily due to neutral protease activity; hyaluronidase does not contribute significantly. Evidence for these conclusions is adduced from electrophoresis of degradation products, from comparison of the proteoglycan-degrading and protease activities of extracts and from the effects of activators and inhibitors. 2. Most of the neutral protease activity is insoluble in buffer of low ionic strength and may thus be separated from small amounts of other, soluble, proteoglycan-degrading enzymes. This ‘insoluble’ protease dissolves in KCl (1 mol/l) and has appreciable solubility even at physiological ionic strength. 3. The protease has optimum activity between pH 7.5 and pH 10, with little or no activity below pH 6.0. 4. The enzyme hydrolyses a number of substrates including elastin and two synthetic substrates for elastase. Hydrolysis of the latter is inhibited by dilutions of synovial fluids and serum and it thus differs from the elastase-like esterase activity of synovial fluid and serum. 5. The ‘insoluble’ enzyme fraction caused loss of staining and release of hexuronate from slices of bovine articular cartilage at salt concentrations near physiological and these effects were almost completely inhibited by cell-free synovial fluids. 6. The properties of the synovial fluid cell neutral protease are compared with those of the similar enzyme activity found in granulocytes from blood and it is concluded that the synovial fluid enzyme is probably derived from granulocytes.

Biochemical Studies of Bacterial Sporulation and Germination: VIII. PATTERNS OF ENZYME DEVELOPMENT DURING GROWTH AND SPORULATION OF BACILLUS SUBTILIS

The levels of eight enzymes from several different metabolic pathways were determined during growth and sporulation, as well as in the mature spore, of Bacillus subtilis. Three different patterns of development were observed. In one, the levels of DNA polymerase, adenylate kinase, and nucleoside phosphorylase closely followed that of total protein throughout growth and sporulation. In a second pattern, arginase, malic dehydrogenase, and ornithine transcarbamylase were undetectable in log phase cells and appeared only after the end of this period. In the third, aspartic transcarbamylase and IMP dehydrogenase were present during exponential phase but disappeared by the time of appearance of refractile spores. The pattern of development of several of these enzymes was different in an asporogenous, protease-negative strain. The susceptibility of certain of these enzymes to protease activity in extracts was observed, suggesting a possible role for protease elaborated during sporulation. In most instances, the specific activities of the enzymes in the spore were similar to their levels in stationary phase cells at the time of forespore formation. Certain enzymes were absent from spores or were present at very low levels. The results are discussed in relation to possible mechanisms for the origin of proteins found in the spore core.