Gelatinolytic Proteinases Research Articles

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Relevance of collagen solubility and gelatinolytic proteinase activity for texture softening in chilled grass carp (Ctenopharyngodon idellus) fillets

SummaryThe aim of the present study was to investigate the underlying mechanisms of softening texture in chilled grass carp filletsunderpinning collagen solubility, gelatinolytic proteinase activity and physicochemical parameters. Acid‐soluble collagen (ASC) and heat soluble collagen (HSC) increased markedly, while a significant decline was detected in total collagen and insoluble collagen (ISC) during the first 3 days of storage, coinciding with a loss of shear force and water‐holding capacity (WHC) (P < 0.05). Moreover, the activity of gelatinolytic proteinases was gradually activated and reached the peak at day 3 (P < 0.05). Pearson coefficient analysis showed that gelatinolytic proteinase activity revealed a significant correlation with collagen solubility. Total collagen, ASC, ISC and HSC were significantly correlated with shear force and WHC. Our study clarified that the increase of collagen solubility by gelatinolytic proteinases played an important role for texture softening in the early stage of chilled grass carp fillets.

PARTIAL CHARACTERIZATION OF GELATINOLYTIC PROTEINASES FROM THE SKELETAL MUSCLE OF GRASS CARP (CTENOPHARYNGODON IDELLUS)

Two gelatin-hydrolyzing proteinases with molecular masses of 85 and 72 kDa in the sarcoplasmic fraction of grass carp muscle were detected using gelatin zymography. The gelatinolytic activity in dark muscle was obviously higher than that in white muscle. Optimum pH and temperature of the two enzymes were around 8.0 and 40C. The proteinase inhibitor leupeptin entirely inhibited GP-I, but E-64 did not inhibit it. Only EDTA completely suppressed GP-II, and Ca2+ is essential for the activity of GP-II. All these facts indicated that GP-I was matrix serine proteinase, and GP-II was matrix metalloproteinase. When grass carp muscle was stored for 15 days at 4C, GP-I and GP-II were detected during the whole stored period. Therefore, the two gelatinolytic proteinases may be proposed to participate in the tenderization of fish muscle during postmortem stage. PRACTICAL APPLICATIONS Gelatinolytic proteinases from grass carp can effectively hydrolyze gelatin. These proteinases may be used to hydrolyze gelatin for peptide that has been added to food products as additive to improve texture. In addition, this study will be significant to learn and comparatively study the properties of gelatinolytic proteinases in fresh fish. Furthermore, this study will surely be beneficial for understanding the mechanism of postmortem tenderization of fish muscle.

GELATINOLYTIC SERINE PROTEINASES FROM THE WING MUSCLE OF RED STINGRAY

ABSTRACT Four gelatinolytic proteinases (A1, 97 kDa; A2, 66 kDa; A3, 30 kDa; A4, 23 kDa) were detected from the red stingray wing muscle by gelatin zymography. Among these proteinases, major gelatinolytic proteinases (A1, A2) were more purified and examined their characters. The optimum pH and temperature for the gelatinolytic activity of A1 and A2 were around pH 7 and pH 8, and maximal activity of A1 is at 30–50C, that of A2 is at 30C. Also, they were demonstrated to hydrolyze the gelatin from red stingray; protein bands were almost completely degraded after 6 h incubation at 37C and pH 7.5. Both enzymes were strongly inhibited by Pefabloc SC, which suggests that they belong to gelatinolytic serine protease. PRACTICAL APPLICATIONS Gelatinolytic enzyme hydrolyzes denatured collagen such as gelatin. The enzymes from red stingray may be utilized to produce gelatin peptide, which has been widely used as additive to enhance the elasticity, consistency and stability of food products. In addition, it is meaningful in that this study will give information to understand the gelatinolytic proteinases in ray species and could lead to a comparative study of the biochemical properties of gelatinolytic proteinases in elasmobranch and many other species.

2‐Hydroxyethyl methacrylate as an inhibitor of matrix metalloproteinase‐2

This study evaluated the effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) on the inhibition of matrix metalloproteinase-2 (MMP-2) in vitro. Mouse gingival explants were cultured overnight in Dulbecco's modified Eagle's minimal essential medium, following which the expression of secreted enzymes was analyzed by gelatin zymography and the effects of different amounts of HEMA on enzyme activity were investigated. The gelatinolytic proteinases present in the conditioned media were characterized as being matrix metalloproteinases (MMPs) by means of specific chemical inhibition. The MMPs present in the conditioned media were identified, using immunoprecipitation, as MMP-2. Three major bands were detected in the zymographic assays and were characterized, according to their respective molecular weights, into the following forms of MMP-2: zymogene (72 kDa), intermediate (66 kDa), and active (62 kDa). All forms of MMP-2 were inhibited by HEMA in a dose-dependent manner, implying that MMP-2 may be inhibited by HEMA in vivo.

Purification and Characterization of Gelatinase-like Proteinases from the Dark Muscle of Common Carp (Cyprinus carpio)

Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.

Inhibition of human pulpal gelatinases (MMP-2 and MMP-9) by zinc oxide cements.

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathological and physiological processes such as, periodontal tissue destruction, root caries, dentin calcification and pulpal inflammation. The aim of this work was to test the effect of zinc released from zinc oxide-eugenol (ZOE) cements, on the activity of the major pulpal gelatinolytic MMPs. Pulpal explants were cultured overnight in Dulbecco's Modified Eagle Medium and the activity of secreted enzymes was analysed by gelatin zymography in buffer conditioned with diverse ZOE cements. Phenanthroline, a zinc chelator, was used to revert the inhibition of MMPs caused by zinc. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. All ZOE cements inhibited MMPs activity, whereas phenanthroline could partially revert the inhibition caused by plain ZOE and Intermediate Restorative Material (IRM).

Specific pro-apoptotic fibronectin fragments modulate proteinase expression in periodontal ligament cells.

During inflammation and periodontal disease, the extra-cellular matrix protein fibronectin (FN) is degraded into fragments whose activities differ from those of the intact molecule. In periodontal ligament cells in vitro, some fragments elevate the expression of matrix metalloproteinases and serine proteinases, while others induce apoptosis through a mechanism regulated by caspase proteases and specific signaling pathways. In this study, we investigated whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of the apoptotic mechanism in periodontal ligament cells. The importance of this study is that a structurally similar fibronectin fragment found in vivo (40-kDa) is associated with periodontal disease status. However, the recombinant V+H- FN protein which is structurally and functionally similar to the in vivo 40-kDa fragment was chosen as the focus of these studies over the in vivo fragment because of the ability to readily produce and purify large quantities of recombinant fragments for detailed studies. Thus, findings from this study relate to our understanding of how the extracellular matrix of the periodontal ligament in an inflamed environment may contribute to periodontal disease and its progression. We used substrate zymography, reverse zymography, proteinase inhibitors, and partial sequencing to investigate whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of this apoptotic mechanism. Incubation with the V+H- fragment reduced the expression of all gelatinolytic proteinases and inhibitors commonly expressed by periodontal ligament cells. In the presence of caspase inhibitors, inhibitors known to suppress apoptosis, however, the reduced proteinase profile was rescued, showing that caspase inhibitors were able to reverse the reduced proteinase profile and indicating that caspase-mediated pathways are pertinent to fibronectin-fragment-mediated matrix metalloproteinase expression. In addition, the V+H- fragment also triggered the expression of a unique high molecular weight gelatinolytic proteinase of approximately 200 kDa. This proteinase was a serine proteinase, whose identity is not known. These findings suggest that matrix-degrading proteinases may be involved in apoptosis as part of a unique mechanism of periodontal tissue breakdown, in which novel proteinases may help execute the dissolution of the extracellular matrix.

Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts

Objectives: The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs. Methods: Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma). Results: ZnSO 4 was a strong inhibitor of MMP-2 (I 50=15 μM) and MMP-9 (I 50=40 μM), whereas CuSO 4, HgSO 4 and SnCl 2 showed less efficient inhibition potential. Significance: Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.

Establishment of a new human urinary bladder mixed carcinoma cell line UMK-1 and its cell-density-dependent secretion of gelatinases and tissue inhibitor of metalloproteinase-1.

A new human carcinoma cell line, UMK-1, was established from primary urinary bladder carcinoma with histological diagnosis of transitional cell carcinoma grade 2, with squamous and adenocarcinomatous differentiation. These histologic features were maintained in the cultured cells and in xenografted tumors in nude mice. Ultrastructurally, the cultured cells were characterized by formation of glandular structure with well-formed junctional complexes and microvilli, intracytoplasmic lumina and tonofilament bundles. Upon continuous propagation in serum-free medium, UMK-1 cells secreted gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 into the conditioned medium. The gelatinolytic activities which were inhibited by EDTA, were composed of a major band at 95 kD and a minor band at 68 kD on gelatin zymography. The measurement of TIMP-1 in the conditioned medium was performed by the one-step sandwich enzyme immunoassay system. The production of gelatinases and TIMP-1 was observed during the late log phase of the cell growth and those productions showed similar kinetics. UMK-1 cells can be used to study the interaction of tumor cell-derived gelatinolytic proteinases and TIMP-1 in cancer invasion and metastasis.

Gelatinolytic and type IV collagenolytic activity in bronchiectasis.

To evaluate the extracellular matrix (ECM) degradation in bronchiectasis (BE), the level of gelatinolytic and type IV collagenolytic activity was analyzed in bronchoalveolar lavage fluid (BALF) by using zymographies. The BALF of patients with bronchiectasis revealed a high gelatinolytic and type IV collagenolytic activity whereas no such activities were detected in BALF of the healthy controls. Furthermore, the level of degradative activity correlated with the severity of disease with a spectrum varying from patients characterized by frequent pneumonia and bronchitis, mucopurulent and purulent sputum production, and saccular changes of bronchi having high activities of both 92-kd and 72-kd gelatinases type IV collagenases (corresponding to the neutrophil type MMP-9 and fibroblast type MMP-2 activities, respectively) to patients having few clinical symptoms and displaying only a weak activity at the 92-kd area. These findings suggest a role for the matrix metalloproteinases MMP-2 (72-kd gelatinase/type IV collagenase) and MMP-9 (92-kd gelatinase/type IV collagenase) in the degradation of ECM of bronchial wall and lung tissue. In addition, severe bronchiectasis was associated with the presence of low-molecular weight gelatinases reflecting in vivo metalloproteinase activation and/or the presence of microbial-derived gelatinolytic proteinases.

Metalloproteinase activities expressed during development and maturation of the rat prostatic complex and seminal vesicles.

The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of Specific Gelatinolytic Proteinases in the Lateral Prostate of Rats by Ectopic Pituitary Grafts1

Activities of plasminogen activator (PA) and gelatinolytic proteinase in the ventral, lateral, and dorsal lobes of the prostate were examined in rats with hyperprolactinemia, which was induced by implantation of ectopic pituitary grafts under the renal capsule. Weight and total DNA content of the lateral lobe increased significantly in rats receiving two pituitary glands as compared with values for the initial untreated group and the muscle-implanted control animals. No change in specific activity of PA was observed in any prostatic lobe for animals of different treatment groups. However, there was a lateral lobe-specific induction of gelatinolytic proteinase activity in pituitary-grafted rats. Two Ca(2+)-independent proteinase activities of approximately 26-28 and 88 kDa, and three calcium-sensitive proteinase activities of 59, 64, and 76 kDa were found in zymograms of lateral prostate extracts of the untreated initial group and the muscle-grafted control. The lateral prostate proteinase pattern of pituitary-grafted animals demonstrated intense activities of approximately 51, 53, 64, 76, and 135 kDa as well as weaker activities of 40, 43, 59, 85, 93, 105, and 125 kDa. Many of these proteinase activities were present in the secretion of the lateral prostate of the untreated initial controls, suggesting that hyperprolactinemia promotes secretion in the lateral prostate. Those proteinase activities that were not in the secretion were probably of intracellular origin and may play a role in cellular remodeling. Castration resulted in a decrease in activities of these proteinases, and this decrease was observed whether the pituitary grafts were retained or removed.

Sequential degradation of interstitial collagen by metalloproteinases extracted from tumors of murine ascites hepatomas.

Gelatinolytic and collagenolytic proteinases were separately isolated by different extraction methods from the mouse ascites hepatoma MH134, and from rat ascites hepatoma AH109A. The activities of two proteinases in each extract showed no significant differences, but after trypsin activation the activities of proteinases from the highly metastatic MH134 were significantly increased compared to the enzyme activities in AH109A, which has low metastatic potential. The total activities of collagenase and gelatinase were increased 7.2- and 5.1-fold; their specific activities were increased 5.2- and 4.8-fold, respectively. Gelatinase and collagenase from MH134 were characterized on gelatin zymography. The gelatinase had a molecular weight of 99 and activation by 4-aminophenylmercuric acetate (APMA) or trypsin resulted in its conversion to 79 or 79-95 kD, respectively. The collagenase revealed a major gelatinolytic band at 89 kD, which was converted to 85 and 70 kD by APMA-activation, and a minor gelatinolytic band at 60 kD. These proteinases could degrade native type I collagen to small fragments in a cooperative manner. Trypsin inhibitor, which affects the trypsin activation of latent gelatinase, was extracted together with gelatinase. The inhibitory activity of the enzyme from AH109A showed a 4.1-fold higher specific activity and 3.7-fold greater total activity than that from MH134. The proteinase(s) capable of activating the gelatinase was also extracted from MH134.

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.

Proteolytic enzymes in human eccrine sweat: a screening study.

Thermally induced human eccrine sweat was collected both by simple scraping (SS) and by a polyethylene sweat collector that covered the Vaseline-coated skin of the back to minimize epidermal contamination (CS, clean sweat). Using synthetic chromogenic Kabi S-series substrates, we demonstrated a variety of proteases in both SS and CS, the former being higher than the latter to varying extents. The higher enzyme activity in SS is mainly due to epidermal contamination because abundant protease activity was eluted from the nonperspiring stripped and unstripped skin in vivo and from the stripped stratum corneum itself. The electrophoretic separation of sweat protein has shown that CS contains at least 7 and SS 15 gelatinolytic proteinases. Although some CS proteinases could be derived from the sweat duct, the sweat secretory coil itself is responsible for at least two proteinases at 78 and 25 kilodaltons. The identity and function of these enzymes remain to be studied.