Properties of renal fructose 1,6 bis-phosphatase of goat

The renal fructose 1,6 bis-phosphatase was partially purified from goat kidney. The purified enzyme showed 10.5 as the pH optimum. The enzyme essentially required Mg2+ for its activity. Heavy metals inhibited enzyme activity. Inhibition was relieved by chelator EDTA and cysteine. These results suggest that renal fructose of goat, too, is sulfhydryl-requiring enzyme.     

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

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.     

化学修饰对嗜水气单胞菌弹性蛋白酶活性及稳定性的影响

用10 ku的截流膜超滤、30%~60%的硫酸铵分级沉淀、凝胶过滤层析和阴离子交换层析,获得纯化的嗜水气单胞菌胞外弹性蛋白酶。SDS-PAGE显示,该酶是分子量约为33 ku的单体蛋白。用活化的右旋糖酐及单甲氧聚乙二醇(MPEG)分别对纯化的嗜水气单胞菌弹性蛋白酶进行化学修饰。修饰后的酶与原酶相比,修饰酶保留了天然酶的活性,两种修饰酶活性均能保留在60%以上,用MPEG修饰酶的保留活性更高(85.5%),而且两种修饰酶在耐热性、耐酸性等方面都优于天然酶,但修饰后酶的最适pH没变。修饰酶较天然酶稳定,具有较高的实用意义。     

产蛋白酶芽孢杆菌的筛选鉴定及酶学特性分析

为了分离筛选产蛋白酶芽孢杆菌,以开发新型饲料添加剂,本研究选取小鼠肠道内容物为样品,通过对样品进行预处理以及酪蛋白平板检测法分离筛选获得产蛋白酶芽孢杆菌,对该菌进行形态学和分子生物学鉴定并分析其生长规律、产酶规律以及所产蛋白酶的酶学特性。结果表明,经形态学和分子生物学鉴定该菌为特基拉芽孢杆菌(Bacillus tequilensis),其所产蛋白酶的最适反应条件为50℃,pH=8.0,具有一定的高温耐受性,在弱酸、中性和弱碱性条件下表现出一定的稳定性,Ca²⁺和Mn²⁺能够显著提高酶活(P<0.05),Zn²⁺、Mg²⁺、Cu²⁺和EDTA、尿素溶液对蛋白酶活力具有显著抑制作用(P<0.05),培养至16 h菌体量达到最大值,发酵22 h上清液中的蛋白酶活力达到最高值(57.77 U/mL)。该芽孢杆菌的成功筛选分离以及其所产蛋白酶的分析研究,为新型饲料添加剂的开发利用提供了物质基础。     

Bovine metalloprotease characterization and in vitro connective tissue degradation

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.     

The purification and characterization of a cysteine protease of Fasciola gigantica adult worms.

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.     

鸡骨、肝和肠硷性磷酸酶的提纯和性质

用正丁醇抽提、DEAE纤维素(DE—52)和Con·A—Sepharase 4B柱层析等方法从鸡的骨、肝和肠组织中提取碱性磷酸酶(ALP)。提纯倍数分别为41倍(骨)、391倍(肝)和130倍(肠)。将粗提的ALP进行聚丙烯酰胺梯度凝胶电泳,骨、肝ALP均出现两条带,其分于量为154 000和353 000(骨),187 000和353 000(肝);肠ALP有3个组分,分子量为123 000、235 000和327 000。骨、肝、肠的ATP经DE—52柱层析均得到两个酶峰,其中总活性较大的峰(主峰)进行Con·A—Sepharose 4B柱层析后,其ALP对磷酸苯二钠的km值分别为0.532mM(骨)、0.452mM(肝)和0.472mM(肠)。骨、肝ALP对热敏感,而肠ALP则比较耐热,56℃作用9min,酶活性降低98.8％(骨)、95％(肝)和59.3％(肠)。尿素可抑制骨和肝ALP,肠ALP对尿素不大敏感。4 mol/L尿素对骨ALP活性抑制100％,对肝ALP 95％,对肠ALP仅为47％。2 mmol/L左旋咪唑能抑制骨ALP活性的57.1％、肝ALP的57.7％、肠ALP的15.7％。20 mmol/L L—苯丙氨酸能抑制肝ALP活性的12％、骨ALP的18.5％、肠ALP的57％。低浓度L—苯丙氨酸(1～15mmol/L)对骨ALP几乎没有抑制作用,而对肝ALP有一定抑制作用。上述结果表明,鸡的骨、肝和肠ALP可分为两型:一型为肠ALP,另一型为骨和肝ALP。     

Comparing two commercial enzymes to estimate in vitro proteolysis of purified or semi-purified proteins

Two experiments compared the suitability of two commercial enzymes to estimate in vitro proteolysis of different proteins. Experiment 1 compared the proteolytic activity over various incubation times of a microbial enzyme (protease from Streptomyces griseus) with a plant enzyme (papain from Papaya latex) by using either 1.33 (high, H) or 0.4 (low, L) units (U, amount) of each enzyme per mg crude protein (CP) of purified proteins including bovine (BA) or egg albumin (EA). Experiment 2 compared the activity of 0.66 U of each of these enzymes per mg CP of semi-purified proteins including casein, wheat gluten (WG) and maize gluten meal (MG). Each incubation was terminated by adding trichloroacetic acid (TCA) and the TCA soluble supernatant collected to estimate concentration of total amino acids (AA) as the measure of proteolysis of each protein over each time. The data on proteolysis over time were fitted into a non-linear model to derive constants for solubility (a) and rate (c) and extents (a + b) of proteolysis of each food by each amount of each enzyme. All data on proteolysis over time and the derived constants were statistically analysed to study the effect of food, enzyme, amount and their interactions. Significant differences were observed between foods, enzymes, enzyme amount (H vs. L for Experiment 1 only) for the proteolysis at most incubation times in both experiments (p<0.001). The mean proteolysis over all times for BA was 1.6 (SD, 0.52) times greater than EA (p<0.001). While the protease gave about four times (SD, 2.1) more proteolysis than papain (p<0.001), the high amount of enzyme gave only about two times (SD, 0.28) greater proteolysis than that of the low amount (p<0.001). On average, the protease was over three times faster (c) than papain (p<0.001) and high amount was two times faster than the low amount of enzyme (p<0.001). While both purified foods were similar in solubility (p>0.05), they differed in the rate and extent of proteolysis (p<0.001). The BA was degraded about four times faster than EA (p < 0.001). Amongst semi-pure proteins, casein gave the highest but MG the lowest proteolysis at each incubation (p<0.001). However, the magnitude of proteolysis depended upon enzyme, food and hours of incubation. On average, casein was degraded at a much faster rate than WG or MG by both enzymes. It appeared that the protease and not papain can be used to estimate in vitro proteolysis of pure and semi-pure food proteins. However, further studies are needed to standardise the relevant procedures when using protease to estimate proteolysis of ruminant foods.     

Cysteine protease in bovine milk capable of hydrolyzing casein as the substrate and elevation of the activity during the course of mastitis

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.     

Investigations about influence of the content of plant crude protein in the ration on the utilization of urea in dairy cattle. 6. Digestive enzymes and their interactions in contents of proximal small intestine

Forty-two samples were taken from the contents of the proximal small intestine of two lactating dairy cows fitted with re-entrant duodenal cannula. Most samples were free of detectable amylase activity. The (chymo)trypsinogen present was only partially activated to (chymo)trypsin. The activation was continued in vitro: slowly at the original pH of the samples (between pH 2.8 and 4.2), and faster after neutralization or a slight alkalinization. The effect of Ca, EDTA and soybean inhibitor on the activation of trypsinogen was also studied. The pancreatic enzymes were inactive in the acid pH range of the samples, but pepsin was markedly active. At pH 3.8 casein was digested rapidly by purified pepsin and slowly by the samples (agar-plate experiments). In model experiments performed with purified enzymes, pepsin digested (chymo)trypsin rapidly at pH 1-2 and slowly at pH 3.8. In the intestinal juice (chymo)trypsin and their zymogens seemed to be unaffected by pepsin under the conditions of the samples. It is concluded that the conditions prevailing in the duodenum/upper jejunum of the experimental cows account for a gastric-type digestion, despite the presence of pancreatic enzymes. In vivo the intestinal contents pass in distal direction. Meanwhile the pH of the chyme gradually increases and gives rise to first an increase of enterokinase activity accounting for a faster activation of the zymogens; second a start of function of activated pancreatic proteases and third a gradual decrease of pepsin activity and finally to its irreversible denaturation. Thus the development of intestinal type digestion is delayed in ruminants.     

Measurement of protease thermostability, twitching motility and colony size of Bacteroides nodosus

Total extracellular protease activity of Bacteroides nodosus in TAS liquid culture varied directly with cell mass and buffer concentration between 20 and 50 mM HEPES, MOPS and TES buffers, but not with Tris which gave anomalous high cell counts, nor with Na2Co3 which showed a decline of protease activity and cell mass. The stability of HEPES-buffered crude protease preparations were estimated on the basis of temperature or Ca2+ activity. Variation of the estimates for cellular twitching was greater than that for colony diameter in benign and virulent strains of B. nodosus. Surface translocation, quantified on the basis of colony diameter, reached a limit after 72 h incubation on modified TAS agar, ranging from 0.04 to 0.14 mm per h for six isolates tested.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

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.     

Enzyme activity in cell cultures infected with herpesvirus suis]

The activity of succinate dehydrogenase (SDH) and lactage dehydrogenase (LDH) was studied in chick-embryo fibroblast cultures after inoculation of the virulent strain "A2" and the avirulent strain "MK" of herpesvirus suum. Strain "A2" reduced SDH activity, and so did strain MK, but here the decrease of enzyme activity was slower, and it did not become evident until the 24th hour. LDH activity fluctuated after "A2" infection but was generally increased, while there was no change in LDH activity, compared with uninfected control cells, after "MK" infection. When interaction of cell and virus took place in the presence of 5-iodo-2-desoxyuridine (IUDR), strain "A2" produced little change in the enzymes, but "MK" infection was accompanied by a definite fall in SDH and a slight increase in LDH. The presence of IUDR inhibited the proliferation of the virulent strain but had no apparent effect on proliferation of the attenuated strain "MK". Investigation of the enzyme activity of cells infected with Aujeszky's disease virus has revealed new biological properties of the virus, which might serve to distinguish between different strains of the virus.     

Influence of dietary phosphorus level on growth performance in chicks given corn-soybean diet supplemented with amylase and acid protease

Two experiments were conducted to determine if dietary amylase and acid protease supplementation improves the nutritive value of corn-soybean diet, and whether the dietary inorganic phosphorus (P) level affects the growth-promoting effect induced by the enzyme supplementation in chicks. In the first experiment, 4-day-old Single Comb White Leghorn male chicks were given a corn-soybean diet supplemented with amylase and acid protease for 10 days. Dietary amylase addition produced a significant improvement in growth and food efficiency, whereas acid protease had no effect on these parameters. Although there was no significant interaction between amylase and acid protease, the combination of the two enzymes produced the greatest improvement in growth performance. In contrast, neither enzyme influenced the metabolizable energy value or nitrogen balance. In the second experiment, chicks were given corn-soybean diets having low, medium and high levels of inorganic P (3.5, 4.3 and 5.1 g/kg, respectively), supplemented with both enzymes. There were significant interactions between the dietary inorganic P level and enzyme supplementation on final bodyweight, bodyweight gain and food intake. Enzyme supplementation significantly improved chick growth at the medium inorganic P level, but not at the low or high levels. Although the thigh bone ash content and serum P concentration were improved as the dietary inorganic P level increased, enzyme supplementation had no influence on these parameters. In conclusion, the present results indicate that dietary amylase and acid protease have beneficial effects on growth performance in chicks given corn-soybean diet. The dietary inorganic P level affects the growth response to dietary amylase and acid protease supplementation.     

大麦芽阿魏酸酯酶的分离纯化及其部分酶学性质的测定

本试验采用硫酸铵分级沉淀、透析、DEAE-52离子交换层析对大麦芽中的阿魏酸酯酶(FAE)进行了分离纯化,并测定了其部分酶学性质。结果显示,分离纯化后获得了阿魏酸酯酶纯品,纯化倍数达34倍;经SDS-PAGE电泳显示为单一条带,并测定其相对分子质量约为29.3 ku;最适反应温度为50℃;最适pH为5.5,但阿魏酸酯酶在60℃以下,pH 4.5~7.5之间有较好的稳定性;Zn2+、Cu2+、Fe3+对酶活有很强的抑制作用,Na+和EDTA对酶有一定的激活作用。     

Quantification of Ca2(+)-dependent protease activities by hydrophobic and ion-exchange chromatography

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.     

人组织蛋白酶K在毕赤酵母中的表达、纯化及活性测定

在动物和人的骨代谢疾病中,组织蛋白酶K是木瓜蛋白酶家族中的一种半胱氨酸蛋白酶,是负责骨吸收过程中溶骨的主要关键酶,是新药物的分子作用靶点。通过毕赤酵母表达系统表达出重组组织蛋白酶K,进一步用离子交换和分子筛系统进行纯化得到高纯度有活性的重组组织蛋白酶K。运用FluStar荧光酶标仪测定390 nm(激发光)和460 nm(发射光)波长下荧光强度的变化并计算出组织蛋白酶K的酶活力为119.6 U,为进一步筛选骨质疏松等骨代谢疾病的新药提供参考。     

添加外源性酶对猪、鸡内源消化酶活性的影响

观察了猪、鸡不同类型饲粮中添加外源性酶制剂后对其内源性消化酶活性的影响。结果,玉米－豆粕型饲粮中添加外源酶后,３７及６７日龄丝毛乌骨鸡的小肠胰蛋白酶活性分别提高１３．１５％（Ｐ＜０．０５）和９．４５％（Ｐ＜０．０５）,总蛋白水解酶活性提高７．３０％（Ｐ＞０．０５）和１３．７％（Ｐ＜０．０５）,但α－淀粉酶活性无显著变化;高麸皮饲粮中添加外源酶可使４９日龄ＡＡ肉鸡的胰蛋白酶,总蛋白水解酶和α－淀粉酶活性分别提高４６．５０％（Ｐ＜０．０５）,１５．２０％（Ｐ＞０．０５）和７９．８０％（Ｐ＜０．０５）,而对胰脏中α－淀粉酶、脂肪酶活性无显著影响;高次粉饲粮中添加外源酶可使杜长大仔猪的小肠总蛋白水解酶活性提高２１．００％（Ｐ＜０．０５）,而对肠α－淀粉酶活性无显著影响;大麦－豆粕型饲粮中添加外源酶可使杜长大肥育猪的肠胰蛋白酶活性下降３６．９０％（Ｐ＜０．０５）,肠脂肪酶活性提高７２．７％（Ｐ＜０．０５）,而对小肠α－淀粉酶和胰脏α－淀粉酶、脂肪酶活性无显著影响。据此认为,外源性酶与畜禽内源性消化酶活性的关系较为复杂,受到动物品种和饲粮类型的影响,尤其是后者。     

Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis

OBJECTIVE: To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers. SAMPLE POPULATION: Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis. PROCEDURE: Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% alpha1-proteinase inhibitor (PI), 0.5% alpha1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples. RESULTS: Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES. CONCLUSIONS AND CLINICAL RELEVANCE: We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses.     

Enzymes of isolated brush border membrane of Cotugnia digonopora, and their insensitivity to anthelmintics in vitro

Purified brush border membrane of Cotugnia digonopora showed the presence of a number of phosphohydrolases. Among these, alkaline phosphatase was extremely active. Other enzymes such as glucose-6-phosphatase, fructose-1,6-diphosphatase, cAMP-phosphodiesterase, 5'-nucleotidase and adenosine-triphosphatase were also active. Observations were made on the activities of various ATPases; whereas the enzyme was activated by Ca++ and Mg++ in an additive manner, its sensitivity to ouabain was negligible. Furthermore, in the presence of EDTA the enzyme activity was quite significant. The treatment of isolated brush border membrane with mebendazole, niclosamide and praziquantel in vitro did not alter the activity of these enzymes. However, treatment of intact worms drastically affected the integrity of the membrane.