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1.
Postmortem proteolysis in skeletal muscle and factors affecting this process were examined in pork, lamb and beef longissimus muscles (LM) to determine the cause of differences in meat tenderness among these species. Fat thickness differed among species in the following order: pork greater than beef greater than lamb. The following patterns were observed for rate of temperature and pH decline: lamb greater than pork greater than beef and pork greater than beef greater than lamb, respectively. At 1 d postmortem, pork was the most tender, followed by beef and lamb, respectively. Between 1 and 14 d of postmortem storage, lamb LM was the most improved in tenderness, followed by beef and pork, respectively. Species did not differ (P greater than .05) in LM collagen solubility. Pork LM from fed pigs had the highest (P less than .05) level of cathepsins B + L and cystatin(s) activities, whereas no differences (P greater than .05) were observed among the species for cathepsin B activity. The lowest (P less than .01) Ca2(+)-dependent protease (CDP)-II and CDP inhibitor activities were observed in pork LM. Beef LM had the highest CDP inhibitor activity (P less than .05) but was intermediate in CDP-II activity. No differences were observed among species for CDP-I activity. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrils isolated at 0, 1 and 14 d postmortem indicated that by d 1, desmin hydrolysis was most extensive in pork muscle, followed by lamb and beef.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
Activities of acidic proteases (cathepsin B + L) and neutral, calcium-dependent proteases (CDP) were quantified to determine whether differences in proteolytic activity could explain differences in meat tenderness among breed types. Steers (n = 32) of known percentage Angus (A) and Brahman (B) breeding were used to establish differences in meat tenderness (A; 3/4A-1/4B; 1/2A-1/2B; 1/4A-3/4B). Samples were removed from the longissimus muscle within 1 h postmortem and within 2 h were frozen for subsequent determination of cathepsin B + L, CDP-I, CDP-II and CDP-inhibitor activities. Warner-Bratzler shear (WBS) was assessed after 1, 5 and 10 d of postmortem aging. Taste panel evaluations, conducted on steaks that were subjected to 5 d of aging, detected no differences. At d 1, WBS did not differ among breed types; however, by d 10 of aging, steaks from Angus steers were more tender (P less than .05) than steaks from 1/2B and 3/4B steers. The Angus and 1/4B steaks had significantly more (P less than .05) cathepsin B + L activity than the 3/4B. The CDP had no relationship with WBS; however, CDP-inhibitor was positively related to d-1 WBS (r = .41, P less than .05). Cathepsin B + L activity was negatively related to WBS at d 10 (r = -.44, P less than .05). These data suggest that differences in meat tenderness among breed types may be explained partially by differences in proteolytic enzyme activity.  相似文献   

3.
An extraction and assay system was developed for quantifying endogenous muscle proteases from a single 5-g sample. A single extraction buffer was developed for simultaneous extraction of both calcium-dependent proteases (CDP) and cathepsins. Protease activity determined by the modified procedure was compared to standard procedures currently used in our laboratory. The successful use of the modified procedure on muscle biopsies was verified. Activities per gram of ovine longissimus muscle of CDP system components for 50-g standard and 5-g modified procedures were not different (P greater than .05) for CDP-I (1.16 vs 1.08), CDP-II (.89 vs 1.03), or CDP inhibitor (2.34 vs 2.32), respectively. Activities of cathepsins per gram of muscle for standard and modified procedures were higher (P less than .05) for the modified procedure (cathepsins B + L, 202.0 vs 309.8), but not different (P greater than .05) for cathepsin B (76.6 vs 98.8). Cystatin-like activity was not different (P greater than .05; 3.4 vs 3.2). To test the effect of location within the longissimus muscle on protease activities, 5 g of longissimus muscle was removed immediately postmortem from each of six locations from each side of three steer carcasses. Location within the longissimus muscle had no effect (P greater than .05) on the protease activities measured. Protease activities determined on bovine longissimus muscle biopsies with the modified procedure were similar to immediate postmortem activities. These data verify that the modified procedure was as able to quantify endogenous muscle proteases as the standard procedures and could be used on muscle biopsies. This procedure should be useful in studying the role of endogenous muscle proteases in muscle growth and postmortem proteolysis.  相似文献   

4.
The objective of this study was to examine the effectiveness of CaCl2 infusion in overcoming the toughness of meat associated with dietary administration of a beta- adrenergic agonist (BAA) to lambs. Thirty-two crossbred (1/2 Finnsheep X 1/4 Dorset X 1/4 Rambouillet) wether lambs were randomly assigned to receive 0 or 4 ppm BAA (L644,969; Merck, Sharpe and Dohme Research Laboratories) in a completely mixed, high-concentrate diet for 6 wk. Animals were slaughtered in two groups of 16. At each slaughter time half of each group (0 or 4 ppm BAA) was randomly assigned to CaCl2 infusion. Feeding the BAA decreased (P less than .05) fat thickness, kidney-pelvic fat, yield grade, and marbling and increased (P less than .05) dressing percentage, lean firmness, leg score, and biceps femoris weight. Weight of biceps femoris was 32.8% greater in BAA-fed lambs. Treated, but not infused, lambs were significantly less tender than control lambs after 1, 7, and 14 d of postmortem storage. At 24 h postmortem, BAA-fed lambs had higher (P less than .05) cathepsin B, calcium-dependent protease-II (CDP-II), and CDP inhibitor activities. Calcium chloride infusion increased marbling, decreased lean firmness, increased lean color score, and increased dressing percentage (P less than .05). Infusion of carcasses with CaCl2 decreased (P less than .05) shear force at all postmortem times. Infusion of carcasses with CaCl2 had no effect on cathepsins B and B + L activities, but it had a significant effect on CDP-I, CDP-II, and CDP inhibitor activities.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.  相似文献   

6.
A 26-28 kDa protease was isolated from Fasciola gigantica adult worms by a two-stage purification process of column chromatography in a Sephacryl S-200 column and affinity chromatography in an L-phenylalanine-agarose column. This protease is a cysteine (thiol) proteinase with an optimum pH of 4.5 and is not inhibited by anti F. gigantica immunoglobulin G. The enzyme was inhibited by protease inhibitors known to inhibit cysteine proteases but not by metallo-, aspartate or serine protease inhibitors. The effect of several protease inhibitors and anti-F, gigantica IgG was also assessed on the total proteolytic activity of F. gigantica. There appears to be a preponderance of cysteine protease activity in F. gigantica and there was a significant inhibition of total proteolytic activity by anti-F. gigantica IgG.  相似文献   

7.
This study identified and characterized hydrolytic enzymes in salivary gland products of Oestrus ovis larvae. Third instars were collected from the heads of slaughtered goats. Salivary glands were extracted, their products obtained by centrifugation and the enzymatic profile determined. Optimum pH, temperature of maximum proteolytic activity, thermal stability, and resistance of salivary gland products were determined on collagen and subclasses of proteases were identified using protease inhibitors. Zymograms were used to determine the molecular weight of proteases. Antigenic protein bands were revealed by immunoblotting using sera obtained from experimentally infested goats. Seven positive enzymatic activities were detected in salivary gland products: acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase (C4), esterase lipase (C8), leucine arylamidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Optimum pH for proteolytic activity was 8.0; proteolytic activity increased with temperature (10-50 degrees C) then drastically decreased at 60 degrees C. Proteases in O. ovis salivary gland products belong to the serine subclass. In Zymograms, bands of proteolytic activity were detected in the 20-63 kDa range; the immunoblot showed three antigenic bands, one of them related to a protease band (63 kDa). Serine proteases in O. ovis salivary gland products are most likely involved in larval nutrition and host immuno-modulation.  相似文献   

8.
A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.  相似文献   

9.
Immature Fasciola hepatica release a papain or cathepsin B-like proteolytic enzyme which cleaves immunoglobulins (Ig) of mouse, rat, rabbit and sheep in vitro. Mouse IgG and IgM molecules are both susceptible to cleavage as is hemoglobin. Whether single or multiple proteases are responsible for Ig cleavage is unknown. The proteolytic activity of secreted enzyme(s) is optimal at pH 3.5-4.5, but activity is also present at pH 7. Proteolysis is enhanced in the presence of 5 mM dithiothreitol or 100 mM cysteine. Based on studies with protease inhibitors, the F. hepatica enzyme activity has been identified as a thiol protease. It is destroyed by heating at 56 degrees C for 1 h, but retains activity after storage at -20 degrees C for 7 days. Whether inhibition of the proteolytic activity increases the susceptibility of F. hepatica immature worm to any extant immune effector mechanisms in hosts remains to be determined.  相似文献   

10.
The purpose of this study was to reveal the possible relationships between total Ca2+-dependent proteinase (CDP) and micromolar-Ca2+-dependent proteinase (microM CDP) activity and cimaterol-induced hypertrophy of skeletal muscle. Dietary administration of 10 ppm cimaterol to finishing lambs reduced microM CDP activity in longissimus muscle (LD) by 55% (P less than .01) and 70% (P less than .02) after 3 and 6 wk of treatment, respectively. Total CDP activity was unaffected by cimaterol at both treatment intervals. The reduced microM CDP activity was not associated with a reduced yield of enzyme extract from the muscle. Cimaterol treatment increased the cross-sectional area of the LD by 23.5% at 3 wk and by 35.6% at 6 wk (P less than .001). Cimaterol also increased (P less than .001) the masses of semitendinosus, semimembranosus and biceps femoris by 26%, 32.4% and 24.5%, respectively. These results suggest that cimaterol-induced muscle hypertrophy may be attained in part by reduction of myofibrillar protein degradation.  相似文献   

11.
Protease enzymes, produced by Bacteroides nodosus strains isolated from animals with virulent and benign forms of ovine footrot, were partially purified by ultra-filtration, ion exchange chromatography and gel permeation chromatography. Each enzyme had a similar pH optimum, was inhibited by phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA) and ethyleneglycot-bis-aminoethylether-N,N-tetraacetic acid (EGTA), but was not inhibited by 1,10-phenanthroline. The results suggest that these enzymes are serine proteases that require divalent cations for activity. The enzymes could be distinguished by their differential temperature stability and differing susceptibility to irreversible inactivation by EDTA. Both enzymes were stabilised by incubation in the presence of Ca2+, but the enzyme purified from the virulent isolate required less Ca2+ for maximum stability. These results suggest that the differential thermostability of the protease activity detected in virulence tests is an intrinsic property of the protease enzymes.  相似文献   

12.
Metalloproteases that selectively hydrolyze connective tissue proteins may tenderize meat without creating texture problems associated with myofibrillar protein degradation. Our objective was to characterize the activity of bovine placental proteases to determine whether they can improve meat tenderness through disruption of the connective tissue matrix. Enzymes were extracted, crudely purified, and proteolytic activity was assessed against gelatin and collagen under varying pH and temperature conditions using both SDS-PAGE and zymography. Gelatin zymography revealed proteolysis between 57 and 63 kDa, with decreased activity as buffer pH decreased from pH 7.4 to 5.4 (37 degrees C). Proteolytic activity was pronounced at 37 degrees C, moderate at 25 degrees C, and absent at 4 degrees C following 48-h incubation (pH 7.4). Placental enzymes were metalloproteases inhibited by excess EDTA. Maximum proteolysis was achieved in the presence of Ca2+, with or without Mg2+ and Zn2+. Absence of Ca2+ decreased proteolytic activity. Complete degradation of both the 125- and 120-kDa proteins of the alpha-chains of gelatin was achieved following enzyme incubation for 6 h at 37 degrees C or 24 h at 25 degrees C. No degradation was observed following enzyme incubation with native Type I collagen. Given the marked decrease in enzyme activity at pH 5.4 and 4 degrees C (standard industry conditions), bovine placental metalloproteases would not be expected to contribute to connective tissue degradation or improve meat tenderness.  相似文献   

13.
Extracellular proteases produced by Bacteroides nodosus in a peptone rich modified trypticase-arginine-serine broth medium were separated and characterised by relative mobility (Rf) in electrophoretic zymogram gels. One benign and two virulent protease banding patterns were established with isolates from sheep, cattle and goats. They correlated with other laboratory tests for virulence but were independent of serogroup. The electrophoretic zymogram method was unable to differentiate intermediate from virulent strains. The time required for the production of maximum levels and numbers of protease bands was four to five days for benign and five to six days for virulent B nodosus. Elevated temperatures (above 45 degrees C) and pH extremes (below pH 6 and above pH 9) modified the electrophoretic banding patterns. The molecular weights of the proteases ranged from 8000 to 43,000 daltons and the isoelectric points from pH 4.90 to 5.90. They are serine proteases and this property can be utilised in affinity purification of these molecules.  相似文献   

14.
1. The role of beta2-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours. 2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective hbetaagonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP. 3. The decrease in the rate of total protein degradation induced by 10(-5)M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5)M ICI 118.551, a selective fbeta2antagonist. 4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4)M dibutyryl-cAMP or isobutylmethylxanthine. 5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by fbeta2adrenoceptors, with the participation of a cAMP-dependent pathway.  相似文献   

15.
The proteases of adult Fasciola gigantica whole worm were analysed by preparative isoelectric focusing and by gelatin-substrate gel electrophoresis at acidic and neutral pH (4.5 and 7.0). At least 15 bands of proteases were observed. These proteases had molecular weights ranging from 26 to 193 kDa and isoelectric points of 4.92–7.63. Potease-rich fractions were subsequently separated from whole worm preparation of the parasite by filtration in Sephacryl S-200. The proteases were able to digest bovine immunoglobulin G (IgG) and globin (derived from bovine haemoglobin) in vitro. The sizes of the proteases in these fractions were from 26 to 96 kDa, and they were inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF), leupeptin and trasylol.  相似文献   

16.
Cysteine protease was found to be present in bovine milk that catalyzed casein as the substrate. The protease was activated by reducing agents such as 2-mercaptoethanol and inhibited by monoiodoacetic acid, but not affected by the addition of phenylmethylsulfonyl fluoride, calcium or ethylene glycol bis (beta-aminoethyl)-N,N,N',N'-tetraacetic acid. The protease activity was linear as a function of protein amount and incubation time, and showed maximum at pH 6.0. By Sephacryl S-200 chromatography, at least two types of cysteine proteases having molecular weights of 45 kDa and more than 150 kDa were detected. The activity was increased in mastitic milk, and well correlated with the stages of mastitis, as indicated by the California mastitis test, somatic cell count and protein concentration. These results suggested that cysteine protease(s) is involved in the pathogenesis of mastitis.  相似文献   

17.
Specific antibodies directed against enterotoxic E. coli strains from the yolk of immunized hens were exposed in vitro to the influence of varying digestive phenomena like reduction of pH and proteolytic digestion. The remaining antibody activity was tested in a specific ELISA system. It could be shown that already within the stomach a considerable loss in antibody activity caused by a lowering of pH and peptic cleavage can be expected. A further loss in antibody activity is due to the proteolytic effect of the pancreas proteases trypsin and chymotrypsin. It was found that antibodies in protein rich solutions like egg or yolk suspensions were more resistant than mere globulin fractions or antibodies isolated by affinity chromatography. Prospects for further in vivo tests are discussed.  相似文献   

18.
Our objective was to determine the predictive value of various biochemical and histological traits for tenderness of the longissimus muscle. Data collected from 27 crossbred cattle included longissimus pH, temperature, sarcomere length, total and percentage of soluble collagen, muscle-fiber type and area, cathepsin B and B + L activities, calcium-dependent protease (CDP)-I, -II and inhibitor activities, myofibril fragmentation indices (MFI), Warner-Bratzler shear (WBS) force, sensory-panel tenderness (SPT) ratings and carcass traits. Stepwise regression analyses were performed among breeds or pooled within breeds with WBS and SPT as dependent variables. When MFI were included in the analysis, MFI at d 7 explained 50% of the variation in WBS and SPT at d 14. An additional 19% of SPT was accounted for by the addition of CDP inhibitor d 1 activity and percentage-area of alpha R fibers to the model. However, because variation in MFI was not significant within breed subclasses and MFI could be classified more as a dependent variable, it was removed from the model. This resulted in CDP inhibitor d 1 activity explaining 44% of the variation in WBS and SPT at d 14. Also, percentage-area of beta R fibers, 6 h pH and cathepsin B + L d 14 activity appeared in the model. In addition, CDP inhibitor activity was the only variable to be significant within breed groups. These data suggest that d 7 MFI could be used as a single predictor of d 14 longissimus muscle tenderness; however, CDP inhibitor d 1 activity (a biological event) also may be useful in predicting tenderness.  相似文献   

19.
《Veterinary microbiology》1998,62(4):321-335
Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7–10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.  相似文献   

20.
Fecal proteolytic activity (FPA) in ten normal dogs was readily detected either calorimetrically using azocasein substrate or by radial enzyme diffusion into agar gels containing casein substrate. Daily FPA ranged from 17 to 207 azocasein units/g (ACUIG) or 4 to 18 mm of casein hydrolysis, while mean 3-day FPA ranged from 58 to 10 1 ACUIG or 7 to 15 mm. Studies of proteolytic activity remaining after treatment of fecal extracts with a specific trypsin inhibitor indicated that trypsin accounted for 0% to 71% of proteolytic activity. Proteolytic activity decreased steadily in fecal specimens stored at room temperature or above, but there was only slight loss in activity during storage for up to 5 days at 4 degrees C. Proteolytic activity was unaffected by repeated freezing and thawing and samples could be stored for long periods at -2 degrees C without noticeable loss of activity. It is concluded that assays of FPA using either azoprotein substrate or radial enzyme diffusion into agar gels containing casein substrate allow evaluation of exocrine pancreatic function in dogs, provided that several samples are tested. These methods are suitable for application in a variety of species.  相似文献   

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