2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: Follow up of cysteine protease isotypes in the course of post-harvest senescence.

Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47–48 kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process

Unveiling a hidden biomarker of inflammation and tumor progression: The 65 kDa isoform of MMP-9 new horizons for therapy

Cancer metastasis is a stage of the disease where therapy is mostly ineffective; hence, the need to find reliable markers of its onset. The metalloproteinase-9 (MMP-9, gelatinase B) in its 82 kDa active form, is a good candidate, but here we show that the correspondent little known 65 kDa active MMP-9 isoform, often misrepresented with the other gelatinase MMP-2, is a more suitable marker. Sera from patients with lung and breast cancer were analyzed by bidimensional zymography to detect the activity of MMP-9 and MMP-2. Enzyme identity was confirmed by comparison with MMP-9 standards and by western blotting. The 65 kDa isoform of MMP-9 is a suitable biomarker to monitor tumor progression from tissue neoplasms to metastatic stage, as its activity begins to appear when disease severity increases and becomes very high in metastasis. Moreover, the 65 kDa MMP-9, which derives from the 82 kDa MMP-9, no longer responds to natural MMP-9 inhibitors. As its activity cannot be controlled, its appearance may warn that the pathological process is becoming irreversible. Identification and inhibition of the enzymes converting the inhibitor-sensitive 82 kDa MMP-9 into the corresponding “wild” 65 kDa MMP-9 may allow to develop therapies capable of blocking metastases

Efficient recovery of whole cell proteins in Oenococcus oeni - a comparison of different extraction protocols for high-throughput malolactic starter applications

In this study, we compared different total protein extraction protocols to achieve highly efficient isolation and purification of total proteins for the specific protein profiling of Oenococcus oeni. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis patterns obtained for the different extraction protocols revealed not only a qualitative similar protein pattern but also quantitative variations with different intensity bands depending on the extraction method used. The selected extraction method added with sonication proved to work extremely well and efficiently and was able to obtain a high resolution 2- D electrophoresis (2-DE) map. Prominent spots were successfully identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and corresponded to 76 different proteins involved in the main metabolic pathways. The approach allowed to achieve a protein profiling specific for O. oeni from Aglianico wine with numerous characterized protein products corresponding to many different O. oeni genes and associated with main cellular pathways. Further investigations of the 2-DE protein expression profile will provide useful and interesting information on the molecular mechanisms at the protein level responsible for growth and survival of O. oeni in wine

Total phenols and flavonoids content, antioxidant capacity and lipase inhibition of root and leaf horseradish (Armoracia rusticana) extracts

Horseradish (Armoracia rusticana Gaertn) is a perennial crop belonging to the Brassicaceae family, widely used as spice in foods and herbal ingredient in ethno-medicine. In this study, were evaluated the phenolic compounds content, antioxidant capacity and anti-lipase activity of methanol, methanol/water (70/30, v/v) and methanol/water (50/50, v/v) extracts of horseradish roots and leaves. Among the extracts tested, both roots and leaves aqueous methanolic (70/30, v/v and 50/50, v/v) extracts showed higher total phenol and flavonoid contents and antioxidant capacity than the corresponding methanol extracts. But extraction yield was high for methanol/water (50/50, v/v) extracts, in both roots and leaves. The extracts exhibited anti-lipase activity in dose-dependent manner. The results showed that the extraction yield and the antioxidant capacity were strictly dependent on the solvent polarity. The results suggest that A. rusticana could provide opportunities for the development of functional food and further in vivo studies for obesity treatment

Digestive Enzymes of the Crustaceans Munida and Their Application in Cheese Manufacturing: A Review

Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20–34 kDa and 4.1–5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40–45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on β-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived β-casein fragment f193–209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet

Analysis of green kiwifruit (Actinidia deliciosa cv. Hayward) proteinases by 2D zymography and direct identification of zymographic spots by mass spectrometry.

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow-up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases formeat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two-dimensional zymography on gelatin-containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI-ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToFMS analysis of the zymographic spots. c 2010 Society of Chemical Industr

The in vitro antioxidant properties of Muscari comosum bulbs and their inhibitory activity on enzymes involved in inflammation, post‐prandial hyperglycemia, and cognitive/neuromuscular functions

Extracts of Muscari comosum bulbs, a traditional Mediterranean food, were analyzed by high performance liquid chromatography for polyphenol content and tested for their activity on free‐radicals and enzymes that might be involved in human health. The extracts revealed the presence of phenolic acids and flavonoids with antioxidant and anti‐inflammatory properties. Antioxidant activity was determined by evaluating the radical scavenging activity toward 2,2‐diphenyl‐1‐picrylhydraziyl (DPPH˙), nitric oxide (˙NO), and superoxide (O2˙−), whereas the anti‐inflammatory activity was determined by zymography by evaluating the in‐gel inhibition of MMP‐9 and MMP‐2, two pro‐inflammatory gelatinases. Anti‐glycemic activity was determined by measuring the inhibition of α‐amylase and α‐glucosidase, two enzymes involved in post‐prandial hyperglycemia. Finally, M. comosum extracts were found to inhibit the enzyme acetylcholine esterase. The resulting increase of acetylcholine availability might improve cognitive functions and neuromuscular transmission. Our laboratory findings substantiate and extend previous results, but the clinical value of M. comosum properties needs to be further evaluated

The Hepatopancreas Enzymes Of The Crustaceans Munida And Their Potential Application In Cheese Biotechnology.

Proteolytic and peptidase activities were extracted from the hepatopancreas of the crustaceans Munida and characterized by enzymatic assay, 2D zymography and mass spectrometry. Results showed the presence of several isotrypsin-like and isochymotrypsin-like enzymes, aminopeptidases and carboxypeptidases A and B. Six different acidic forms of trypsin were detected using specific inhibitors and 2D zymography. Trypsin-like activity was higher than chymotrypsin-like activity. On the basis of previous evidences in food biotechnology and cheese production, the digestive enzymes of the crustaceans Munida were tested for their ability to degrade casein, a process involved in cheese production. As a result, the Munida enzymes were found to degrade the chymosin-derived b-casein fragment f193e209, one of the peptides associated with bitterness in cheese, revealing their possible application in cheese technology to lower the unpleasant bitter flavour in some cheeses