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1.
Background: The concentration of canine adrenocorticotropic hormone (ACTH) is usually determined by radioimmunoassay. However, chemiluminescent assay techniques have many advantages for clinical endocrine testing. Objectives: The objectives of this study were to validate a commercially available chemiluminescent assay for determination of canine ACTH concentration and to determine whether protease inhibitors are appropriate for use in the chemiluminescent assay system. Methods: Biological specificity was evaluated by treatment of 3 dogs with ovine corticotropin‐releasing hormone (CRH) followed by serial measurements of ACTH and by comparison with a previously validated immunoradiometric assay. All samples were collected both in the presence and absence of aprotinin, a protease inhibitor. The assay was further evaluated by measurement of intra‐assay precision, interassay precision, and recovery after dilution. Results: Baseline ACTH concentrations ranged from 5.6 to 15.3 pg/mL, and maximum ACTH concentrations of 158 to 1240 pg/mL were observed 30–60 minutes after CRH administration. Plasma samples collected with aprotinin had significantly lower ACTH concentrations than did samples collected without aprotinin. The intra‐assay coefficients of variance (CVs) ranged from 4.1 to 8.2%, and interassay CVs ranged from 4.6 to 14.8%. Recovery after dilution with canine plasma ranged from 93.4 to 103.0% of predicted concentration; however, inadequate recovery was observed with other diluents. There was a high correlation with the immunoradiometric assay (r= .925) but a significant negative bias (‐32.9, 95% confidence interval ?50.8 to ?14.9). Conclusions: This chemiluminescent assay is a valid technique for measurement of ACTH in canine plasma. ACTH concentration measured by chemiluminescence is lower than that measured by immunoradiometry. Aprotinin decreases the measured concentration of ACTH, and this effect should be taken into account when interpreting results. Diluents supplied with the kit should not be used for dilution of canine samples.  相似文献   

2.
Acrosomal proteases allow the spermatozoon not only to cross the cumulus cells and penetrate the zona pellucida of the oocyte, but also they are needed for the acrosome reaction process (AR). The present study evaluated in vitro the role of trypsin and chymotrypsin in the acrosome reaction of canine spermatozoa by means of protease inhibitors. Spermatozoa obtained from the second fraction of the ejaculate and devoid of seminal plasma were re‐suspended in canine capacitation medium (CCM) and incubated at 38.5°C in 5% CO2. After 2 h (period of sperm capacitation), aliquots of sperm suspension were incubated separately with trypsin inhibitor NPGB (p‐nitrophenyl‐p′‐guanidino‐benzoate); TI (Trypsin inhibitor I‐S Type from soybean) and with chymotrypsin inhibitor TPCK (N‐tosyl‐L‐phenylalanine‐chloromethyl‐ketone) for 30 min. The AR was induced with progesterone and evaluated using the dual fluorescent staining technique ‘Hoechst and chlortetracycline’. Acrosomal exocytosis levels were statistically significant higher in the samples treated with progesterone than in the control without inducer. However, the trypsin inhibitors NPGB, TI and the chymotrypsin inhibitor TPCK reduced the percentage of AR when compared with the control with progesterone and without inhibitor (p < 0.001), where the AR values were 45.63 ± 3.8%, 51.63 ± 2.8%, 58.38 ±4.1% and 71.25 ± 4.9%, respectively. These results show that trypsin and chymotrypsin inhibitors are effective in blocking the acrosome reaction induced by progesterone in canine; in addition, they suggest the participation of respective proteases in the AR process in this species.  相似文献   

3.
In this study the influence of bovine serum protease inhibitors, trypsin and proteases produced by different types of Clostridium botulinum has been investigated. Trypsin and botulinum proteases had the capability of increasing the toxicity in growing cultures in Clostridium botulinum types A, B and E. Trypsin increased the toxin level to a greater extent than proteases from Clostridium botulinum types A, B, C and F. Protease inhibitors did not influence the toxin formation to any extent compared with the controls. The combined effects of proteases and protease inhibitors on the development of toxin in Clostridium botulinum type B were also investigated by adding proteases and protease inhibitors to the same culture at different time intervals. Protease inhibitors did not reduce the toxicity of the cultures as compared to the controls. Altogether a complex relationship seems to exist between protoxin, toxin, proteases and inhibitors in the culture, and the order and time sequence of addition seem to be of importance. The results obtained in this investigation indicate that proteases of Clostridium botulinum play a part in the formation and/or activation of toxin in growing cultures of proteolytic strains such as Clostridium botulinum types A and B. As to the activation of protoxin and progenitor toxin produced by non-proteolytic Clostridium botulinum types B and E, botulinum proteases showed a marked capability of increasing the toxicity in these cultures. Trypsinization may be valuable for the detection of Clostridium botulinum types A and B in foods, as well as for type E, where it is commonly used.  相似文献   

4.
BACKGROUND: Unfractionated heparin (UFH) has a complex pharmacologic profile that necessitates patient monitoring to prevent inadequate anticoagulation or overdosage and hemorrhage. Factor Xa inhibitory assays (to measure anti-Xa activity) are used to adjust UFH dosage and define safe and effective regimens for specific thrombotic disorders in humans. OBJECTIVE: In this study, the accuracy, linearity, and clinical utility of a chromogenic assay were assessed for monitoring UFH anti-Xa activity in canine plasma samples. METHODS: A commercial assay (Rotachrom Heparin, Diagnostica Stago, Parsippany, NJ, USA) was used to measure anti-Xa activity in canine plasma samples spiked with different concentrations of UFH. Background absorbance and assay linearity were compared for canine and human plasmas. Percentage recovery of UFH anti-Xa activity and intra- and interassay imprecisions were investigated by multiple measurements of canine plasma to which known amounts of UFH were added. The spiked plasma samples also were used to determine the heparin sensitivity of an activated partial thromboplastin time (aPTT) test. RESULTS: Canine plasma samples were assayed at a higher dilution than were human plasma samples (3:8 versus 4:8) to eliminate higher background anti-Xa activity in canine plasma. Using this modification, the recovery of anti-Xa activity in canine plasma was linear (R2 > .9) at concentrations of 0 - 0.75 U/mL UFH. Intra- and interassay imprecisions for plasma samples containing 0.5 U/mL UFH were <10%, whereas samples containing 0.25 U/mL UFH had imprecisions of 13% and 24%, respectively. The anti-Xa activity range of 0.5 - 0.75 U/mL caused prolongation of aPTTs to 1.5 - 2.5 times the assay mean. CONCLUSION: Plasma anti-Xa activity of dogs treated with UFH can be accurately monitored using this commercially available chromogenic assay.  相似文献   

5.
Trypsin-like immunoreactivity and total alpha-macroglobulin levels were assessed in serum and plasma samples taken at presentation from 60 cases of spontaneous canine acute pancreatitis of varying clinical severity. Total alpha-macroglobulin was significantly decreased in all severity groups when compared to 119 healthy controls, however there were no significant differences between severity groups. Trypsin-like immunoreactivity was significantly elevated above assay reference range in all groups. Trypsin-like immunoreactivity was significantly elevated in dogs with severe disease when compared to those with mild disease. These results suggest that zymogen release and protease activation, while components of the pathology of spontaneous canine acute pancreatitis, are not directly associated with the onset of multiple organ failure seen in the most severe cases.  相似文献   

6.
A commercially available radioimmunoassay (RIA) kit for measurement of human adrenocorticotropin (hACTH) was validated for use in dogs. Assay sensitivity was 3 pg/ml. Intra-assay coefficient of variation (x 100; CV) for 3 canine plasma pools was 3.0 (mean +/- SD, 33 +/- 0.99 pg/ml), 4.2 (71 +/- 2.4 pg/ml) and 3.7 (145 +/- 3.7 pg/ml) %. Interassay CV for 2 plasma pools measured in 6 assays was 9.8 (37 +/- 3.6 pg/ml) and 4.4 (76 +/- 3.4 pg/ml) %, respectively. Dilutional parallelism was documented by assaying 2 pools of canine plasma at 3 dilutions and correcting the measured result for dilution. Corrected mean concentrations for the first pool were 33 (+/- 0.99), 36 (+/- 4.3), and 33 (+/- 6.8) pg/ml; corrected mean concentrations for the second pool were 145 (+/- 5.4), 141 (+/- 10.8) and 125 (+/- 3.4) pg/ml. Recovery of 1-39hACTH added to canine plasma (6.25, 12.5, 25.0, 50.0, and 100.0 pg/ml) was linear and quantitative (slope = 0.890, R2 = 0.961). To test whether anticoagulant or the protease inhibitor, aprotinin, influences ACTH concentration in canine plasma, ACTH was measured in canine blood collected in 4 tubes containing anticoagulant: heparin (H), heparin + 500 kallikrein inhibitor units (KIU) of aprotinin/ml (HA), EDTA (E), and EDTA + aprotinin (EA). Plasma ACTH concentration was the same when samples containing H and HA, or HA and E were compared, and was significantly (P less than 0.01) lower in samples containing EA.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
OBJECTIVE: To test a modified saline (0.9% NaCl) solution joint washing (lavage) technique that includes the use of vitamin B12 as an internal marker for the evaluation of synovial fluid dilution in lavage samples from canine joints. SAMPLE POPULATION: 9 plasma samples obtained from blood samples of 9 healthy dogs and 9 synovial fluid samples aspirated from stifle joints of 9 cadaveric dogs. PROCEDURE: Photometric absorbances of 25% vitamin B12 solution, canine synovial fluid, and canine plasma were measured in a spectrophotometer to establish an optimal wavelength for analysis. Canine synovial fluid and plasma samples were mixed with the 25% vitamin B12 solution to obtain 1%, 3%, 5%, 10%, 20%, and 50% solutions of synovial fluid or plasma. Diluted synovial fluid and plasma samples were used to simulate joint lavage samples and to examine the possible interference of these substances (synovial fluid or plasma) with the absorbance of the 25% vitamin B12 solution in photometric analysis. RESULTS: The optimal wavelength was found to be at 550 nm. Canine synovial fluid and plasma samples did not interfere with the absorbance measurements of the 25% vitamin B12 solution up to a 50% dilution of plasma or synovial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The modified saline solution joint lavage method with the use of a 25% vitamin B12 solution as an internal standard provides an accurate and reliable technique for the evaluation of synovial fluid dilution in lavage samples from canine joints.  相似文献   

8.
OBJECTIVE: To detect monocarboxylate transporters (MCTs) in canine RBC membranes and to determine the distribution of lactate between plasma and RBCs. SAMPLE POPULATION: Blood samples obtained from 6 purpose-bred Beagles. PROCEDURES: Monocarboxylate transporter isoforms 1, 2, 4, 6, 7, and 8 and CD147 were evaluated in canine RBCs by use of western blot analysis. Lactate influx into RBCs was measured as incorporation of radioactive lactate. RESULTS: 2 MCT isoforms, MCT1 and MCT7, were detected in canine RBC membranes on western blot analysis, whereas anti-MCT2, anti-MCT4, anti-MCT6, and anti-MCT8 antibodies resulted in no signal. No correlation was found between the amount of MCT1 or MCT7 and lactate transport activity, but the ancillary protein CD147 that is needed for the activity of MCT1 had a positive linear correlation with the rate of lactate influx. The apparent Michael is constant for the lactate influx in canine RBCs was 8.8 +/- 0.9mM. Results of in vitro incubation studies revealed that at lactate concentrations of 5 to 15mM, equilibrium of lactate was rapidly obtained between plasma and RBCs. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicated that at least half of the lactate transport in canine RBCs occurs via MCT1, whereas MCT7 may be responsible for the rest, although an additional transporter was not ruled out. For practical purposes, the rapid equilibration of lactate between plasma and RBCs indicated that blood lactate concentrations may be estimated from plasma lactate concentrations.  相似文献   

9.
Canine and human platelets (washed 4 times in a solution containing EDTA, prostaglandin E1, and theophylline to prevent release of alpha-granule constituents) were lysed by being frozen and thawed in the presence of detergent. Radioelectroimmunoassay for von Willebrand factor (vWf) in 5 human platelet lysates produced precipitin rockets, shaped like those produced from vWf in plasma from healthy human beings, and indicated that the mean von Willebrand factor antigen (vWf:Ag) content in platelets from healthy human being was 526 +/- 87 human U/10(12) platelets. Radioelectroimmunoassay for vWf in platelet lysates from 17 healthy dogs with normal plasma. vWf:Ag concentration produced precipitin rockets that looked different from those produced from canine plasma and indicated vWf:Ag content of 59 +/- 35 canine U/10(12) platelets. Inclusion of protease inhibitors in the lysing solution did not normalize the appearance of the precipitin rockets or substantially alter the measured platelet content of vWf:Ag. The array of vWf multimers revealed by sodium dodecyl sulfate-agarose gel electrophoresis of canine platelet lysates had a distinct appearance that differed from that of vWf in canine or human plasma and platelets; the intensity of the canine platelet vWf multimer bands was skewed, with relatively greater density in the lower molecular weight region and faint or undetectable multimer bands in the higher molecular weight region. Electrophoretograms with visible multimers in the high molecular weight region had vWf components that had higher molecular weight than did any vWf components in canine plasma. Radioelectroimmunoassay for fibronectin in these same canine platelet lysates indicated that the fibronectin content in platelets was 2.89 +/- 1.10 mg/10(12) platelets.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Veterinary diagnostic endocrinology laboratories frequently receive hemolyzed plasma, serum, or blood samples for hormone analyses. However, except for the previously reported harm done by hemolysis to canine insulin, effects of hemolysis on quantification of other clinically important hormones are unknown. Therefore, these studies were designed to evaluate effects of hemolysis on radioimmunoassay of thyroxine, 3,5,3'-triiodothyronine, progesterone, testosterone, estradiol, cortisol, and insulin in equine, bovine, and canine plasma. In the first experiment, hormones were measured in plasma obtained from hemolyzed blood that had been stored for 18 hours. Blood samples were drawn from pregnant cows, male and diestrous female dogs, and male and pregnant female horses. Each sample was divided into 2 equal portions. One portion was ejected 4 times with a syringe through a 20-gauge (dogs, horses) or 22-gauge (cows) hypodermic needle to induce variable degrees of hemolysis. Two subsamples of the blood were taken before the first and after the first, second, and fourth ejections. One subsample of each pair was stored at 2 to 4 C and the other was stored at 20 to 22 C for 18 to 22 hours before plasma was recovered and stored at -20 C. The second portion of blood from each animal was centrifuged after collection; plasma was recovered and treated similarly as was blood. Concentrations of thyroxine in equine plasma, of 3,5,3'-triiodothyronine, estradiol, and testosterone in equine and canine plasma, and of cortisol in equine plasma were not affected by hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
Semen samples were collected at weekly intervals for six weeks from eight sexually mature beagles previously shown to produce normal ejaculates. Seminal plasma and sperm fractions were separated by centrifugation and the sodium, potassium, alanine and aspartate aminotransferases, acid and alkaline phosphatase concentrations in the two fractions determined. Regression analysis of the mean weekly values obtained from physical and biochemical examination of the ejaculates showed that sodium ion concentration was highest in seminal plasma. The highest levels of aminotransferases were found in sperm fractions. Those enzymes may be indices of abnormal or damaged spermatozoa. Acid and alkaline phosphatase activity was 100 times greater in seminal plasma than in sperm fractions. Phosphatase concentrations are likely to be dependent on prostate activity. Measurement of acid phosphatase in canine semen therefore may be a useful index of prostate function. The motility of the semen samples was independent of the potassium concentration in seminal plasma. However, there was some evidence of a correlation between sperm motility and the enzyme and sodium content of seminal plasma.  相似文献   

12.
Parathyroid hormone-related protein (PTHrP) was investigated in a canine lymphoma case with hypercalcemia by means of immunoradiomentric assay (IRMA) and molecular analysis. The plasma calcium level of the patient dog was 13.7 mg/dl. The PTHrP concentration examined by IRMA was 6.1 pmol/L in the plasma sample from the dog, but it was undetectable (< 1.1 pmol/L) in plasma samples from 4 lymphoma cases without hypercalcemia or 5 normal dogs. The PTHrP concentration examined in the culture supernatant of the lymphoma cells from this case was 1.3 pmol/L, whereas those of the lymphoma cells from a lymphoma case without hypercalcemia was undetectable. PTHrP mRNA was clearly detected not only in the lymphoma cells from this dog with hypercalcemia but also in lymphoma cells from 4 lymphoma cases without hypercalcemia and 2 canine lymphoma cell lines.  相似文献   

13.
Canine serum alpha 1-proteinase inhibitor is not a major acute phase reactant in the dog, unlike the equivalent protein in humans. The possibility that an alternative protease inhibitor system is stimulated during the acute phase response in the dog was investigated. alpha 2-macroglobulin was not an acute phase reactant, but an inhibitor of elastase was identified in canine serum which could be separated from proteinase inhibitor by gel filtration and which was shown to be an acute phase reactant. This protein has been named canine elastase inhibitor.  相似文献   

14.
OBJECTIVE: To optimize methods used to measure coagulation factor activities in canine plasma, define reference ranges in dogs, and compare activities between canine and human plasma. SAMPLE POPULATION: Human plasma samples (n = 5) and plasma from healthy dogs (140) and dogs with low factor V activity (7), high factor V activity (7), and low factor VIII:C activity (6). PROCEDURE: Coagulometric tests incorporated human plasma deficient in a single coagulation factor (human deficient plasma). Standard curves were generated with pooled plasma from 100 healthy dogs. Effect of sample dilution was evaluated, using plasma from dogs with high or low factor V activity and low factor VIII:C activity. Reference ranges for healthy dogs were established. Activities in human plasma were determined by comparison with standard curves obtained with canine plasma. RESULTS: Activities of factors V and VIII:C in samples diluted < or = 1:20 influenced results of tests for other coagulation factors. Activities of factors V and VIII:C in human plasma were significantly less than in canine plasma. For the other coagulation factors, significant differences in human plasma-to-canine plasma activity ratios were detected among different sample dilutions. CONCLUSIONS AND CLINICAL RELEVANCE: Accurate measurement of coagulation factor activities in canine plasma, using human deficient plasma, requires higher sample dilutions (ie, > 1:20) than typically used for human plasma. Differences in activities between human and canine plasma and nonparallelism of the standard curves emphasize the necessity for use of species-specific standard curves for accurate determination of coagulation factor activity.  相似文献   

15.
Miniature Schnauzers are the first canine breed, in the United States, reported to suffer from primary hyperlipidemia, but this has yet to be documented in other regions. Using over 900 canine plasma samples collected from over seven different veterinary clinics across Japan, the aim of this study was to compare plasma triglyceride (TG) and cholesterol concentrations between Miniature Schnauzers and other purebreeds in Japan. In addition, we investigated the influence of aging and sex on changes to hyperlipidemia incidence in purebred dogs. Our results indicated that both Miniature Schnauzers and Shetland sheepdogs in Japan exhibited remarkably high concentrations of plasma TG and total cholesterol, which are considered to be signs of hyperlipidemia, as compared to other purebred and mixed (Mongrel) canine breeds. Interestingly, the cause and conditions of primary hyperlipidemia in Miniature Schnauzers and Shetland sheepdogs might be different, with hypertriglyceridemia predominantly occurring with Miniature Schnauzers and hypercholesterolemia occurring in Shetland sheepdogs. However, with the influence of aging, the hyperlipidemia evolves into both hypercholesterolemia and hypertriglyceridemia in both groups indicating that the severity of hyperlipidemia positively correlates with aging. Gender differences were also observed with regards to severity. In fact, a higher severity was prevalent with female Miniature Schnauzers than their male counterparts whereas it was more balanced between genders for Shetland sheepdogs.  相似文献   

16.
The susceptibility of adrenocorticotropin (ACTH) in canine blood and plasma to enzymatic degradation has limited the availability of endogenous ACTH assay for veterinary use. This study examined if a proteinase (enzyme) inhibitor, aprotinin, mixed with blood at the time of collection, would limit the loss of immunoreactive (IR) ACTH from canine plasma stored at various temperatures. Blood was collected from laboratory-maintained dogs or dogs with hyperadrenocorticism and placed into EDTA-containing tubes in the presence or absence of aprotinin. Plasma obtained was stored for 4 d at temperatures ranging from −86° C to room temperature (22° C). Results showed that addition of aprotinin preserved IR-ACTH concentrations in plasma stored for 4 d at temperatures ≤ 4° C, or in unfrozen plasma stored inside insulated shipping containers containing frozen refrigerant packs. Plasma collected with aprotinin and stored at 22° C showed a slight (17–23%) but significant (P < 0.05) decline in IR-ACTH. Unfrozen plasma collected without aprotinin showed significant (P < 0.05) loss of IR-ACTH during storage under identical conditions. These data indicate that aprotinin has a profound preservative effect upon canine plasma IR-ACTH and that it may be possible to submit unfrozen samples collected with this inhibitor to appropriate reference laboratories for analysis of IR-ACTH.  相似文献   

17.
OBJECTIVE: To determine whether cross-reactivity exists between canine chromogranin A (CgA) and anti-human CgA antibody and investigate the usefulness of plasma CgA concentration measurements as an index of acute stress responses in dogs. ANIMALS: 12 healthy Beagles. PROCEDURE: Canine CgA was extracted and purified from canine adrenal glands of cadaver dogs for studying cross-reactivity with anti-human CgA antibody. Western blotting with anti-human CgA antibody was performed. Blood samples were collected from dogs at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV administration of saline (0.9% NaCl) solution or insulin. Canine plasma CgA concentrations were determined by use of a CgA ELISA kit with rabbit antiserum against the carboxy-terminal fragment of human CgA. Plasma cortisol and catecholamine (ie, norepinephrine and epinephrine) concentrations were measured by use of an ELISA and a high-performance liquid chromatography method, respectively. RESULTS: Purified canine CgA was specifically detected by use of western blot analysis and an ELISA with anti-human CgA antibody. An increase in plasma CgA concentrations was observed in insulin-induced hypoglycemic dogs. Changes in plasma CgA concentration were correlated with changes in plasma cortisol or catecholamine concentrations of hypoglycemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CgA ELISA kit for determination of human plasma CgA concentrations is applicable to the measurement of canine plasma CgA concentrations. Canine plasma CgA concentrations, along with measurements of plasma cortisol and catecholamine concentrations, correctly reflect insulin-induced hypoglycemic stressed conditions in dogs. Measurement of canine plasma CgA concentrations may provide a useful index for evaluation of an acute stress response.  相似文献   

18.
Background: In routine canine medicine, anticoagulated blood is often the only sample sent to laboratories for diagnostic purposes. This hampers the interpretation of protein electrophoretic tracings because plasma contains fibrinogen, which migrates in the β–γ region. In human medicine, fibrinogen can be precipitated from plasma using ethanol. Objectives: The purpose of this study was to assess ethanol precipitation as a method for removing fibrinogen from canine plasma so as to facilitate the interpretation of electorphoresis results. Methods: Blood samples collected from 40 dogs were divided into plain tubes and tubes containing EDTA (n=20) or lithium–heparin (n=20). An aliquot of plasma from each sample was incubated with ethanol at a final concentration of 100 mL/L. Cellulose acetate electrophoresis was then performed on serum, plasma, and plasma treated with ethanol. To verify the efficiency of ethanol treatment, fibrinogen was added to 5 canine serum samples at final concentrations of 2.5, 5.0, and 10.0 g/L, and electrophoresis was performed before and after ethanol treatment. Results: Visual analysis of electrophoretograms from ethanol‐treated samples confirmed the disappearance of the fibrinogen peak from the β2‐globulin region. Treatment with ethanol caused a significant decrease in the percentage of β2‐globulins and a significant increase in the percentage of α2‐globulins. Absolute values of most electrophoretic fractions were significantly decreased in ethanol‐treated plasma compared with serum. Conclusions: Ethanol treatment successfully removed fibrinogen from canine plasma and normalized electrophoretic profiles, but probably also precipitated proteins other than fibrinogen. Ethanol treatment is recommended to facilitate visual identification of abnormal monoclonal peaks, but not for determining absolute protein concentrations in electrophoretic fractions.  相似文献   

19.
Hydrophobic and ion-exchange chromatography were compared for yield of Ca2(+)-dependent proteases and their inhibitor in studies designed to quantify Ca2(+)-dependent proteases activity for comparative purposes. Ion-exchange (DEAE-Sephacel) proved superior to hydrophobic chromatography (Phenyl-Sepharose). Under the proper conditions, DEAE-Sephacel effectively separated low-calcium-requiring form of Ca2(+)-dependent protease (CDP-I) and CDP inhibitor. Characterization of the assay system for components of the Ca2(+)-dependent proteolytic system separated by ion-exchange chromatography indicated that proteolytic degradation of casein by Ca2(+)-dependent proteases was linear with time for up to 60 min at 25 degrees C and that it was linear up to .4 to .45 units of activity. Therefore, we recommend that, after identification of fractions containing Ca2(+)-dependent protease (CDP-I or CDP-II), these fractions be pooled, and reassayed at a volume that yields values of less than .45 units of activity. Unlike CDP-I and CDP-II, CDP inhibitor lost its activity rapidly with frozen storage (frozen in liquid nitrogen, then stored at -70 degrees C); therefore, inhibitor should be assayed in fresh (unfrozen) samples only.  相似文献   

20.
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   

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