Enhanced Release of Cholecystokinin by Soya-Bean Trypsin Inhibitor in Chickens

Whether ingestion of soya-bean trypsin inhibitor (SBTI) alters the release of cholecystokinin (CCK) was investigated in chickens. A meal of an adequate diet supplemented with SBTI (0, 100, or 1000 mg/kg) was given through a stomach tube, followed by CCK determination with specific CCK-8 antibody. The plasma CCK level increased from a basal level (control diet) of 9.6 ± 0.6 to 13.4 ± 0.6 and 18.1 ± 0.8 fmol/ml plasma at 90 min after feeding the diet supplemented with 100 and 1000 mg SBTI, respectively. Since the SBTI supplementation did not affect crop emptying rates significantly, it was concluded that SBTI by itself enhanced CCK release into circulation in a dose-dependent fashion.     

壳聚糖固定胰蛋白酶的研究

目的：从蟹壳中提取壳聚糖，将胰蛋白酶固定在从蟹壳中提取的壳聚糖上。方法：以自制壳聚糖为载体，戊二醛为双官能团交联剂，将胰蛋白酶固定化。结果：０．２ｇ壳聚糖（干）与４％戊二醛交联后再与５．０ｍｇ胰蛋白酶固定结合，效果较好。固定化酶的表现米氏常数Ｋ′ｍ＝１．５ｍｍｏｌ／Ｌ，而天然酶Ｋｍ＝７．６ｍｍｏｌ／Ｌ；固定化酶最适温度为８０℃，比天然酶提高了２０℃；固定化酶最适ｐＨ为７．０．而天然酶为８．０。该载体来源广，易提取，固定化酶的贮存稳定性很好，２个多月重复使用，酶活力未见明显下降     

TRYPSIN INDUCED VON WILLEBRAND FACTOR RELEASE FROM HUMAN ENDOTHELIAL CELLS IS MEDIATED BY PAR-2 ACTIVATION

Nystedt and co-workers cloned in 1994 a second protease activatable receptor (PAR-2) that could be activated by trypsin but not by thrombin [1]. In this study, we investigated whether trypsin induced stimulation of endothelial cells is linked to PAR-2 activation. We have found by mRNA analysis that endothelial cells of venous and arterial origin express both protease activatable receptors. The functional thrombin receptor and the protease activated receptor-2 (PAR-2) mediate apparently the same effects in human vascular endothelial cells. Both, the activation of the thrombin receptor with thrombin or SFLLRN and the activation of the PAR-2 with trypsin or SLIGRL induced intracellular calcium mobilisation and a subsequent release of von Willebrand factor (vWf) from Weibel-Palade bodies. As a consequence, it can be concluded that endothelial cells have two different receptors mediating the same cellular responses after activation. Copyright © 1996 Elsevier Science Ltd     

蜗牛酶在聚(N-异丙基丙烯酰胺)中的固定化及应用

将蜗牛酶固定到聚(N—异丙基丙烯酰胺)上得到一种热可逆的固定化酶。研究了固定化酶的性能和对几种不同底物的作用情况。结果表明蜗中酶对羧甲基纤维素钠、甲壳胺和可溶性棉花都有较好的降解活性。     

Elevated serum levels of immunoreactive anionic trypsin (but not cationic trypsin) signals pancreatic disease

Summary Pancreatic juice from most studied species contains two major forms of trypsin, one with anionic electrophoretic mobility and one with cationic mobility. They are referred to as anionic and cationic trypsin(ogen). The purpose of this study was to measure immunoreactive anionic trypsin (irAT) and immunoreactive cationic trypsin (irCT) in sera from patients with pancreatic cancer (n=39) and chronic pancreatitis (n=32) using two specific ELISA methods. Sera from 72 healthy persons were used as controls. Patients with pancreatic cancer showed significantly elevated serum levels of irAT median level 39 vs 20.5 μg/L in the control group (p<0.001). No differences in irCT levels were found. The ratio between irAT and irCT in serum was significantly increased (p<0.001). Patients with chronic pancreatitis showed a wide range of both irAT and irCT levels, but no significant differences compared to the control group. The ratio between irAT and irCT was, however, significantly increased also in this group of patients. The results suggest a nonparallel secretion of anionic and cationic trypsinogen in pancreatic disease. This is a pattern that has been observed in experimental forms of chronic “hyperCCKemia.”     

甲壳糖固定血管紧张素转换酶的制备及其性质

目的 将血管紧张素转换酶(ACE)固定化并测定其动力学性质。方法 以甲壳糖为载体，碳二亚胺(EDC)为交联剂，与分离纯化的ACE混合,使ACE固定化；并以马尿酰甘氯酰甘氨酸(HCG)为底物，分别测定了其游离酶和固定化酶的动力学性质。结果 固定化酶活力110u／g湿重，固定化率为22％，与游离酶比较，固定化酶稳定且具良好的耐热性，在50—80℃固定化酶较游离酶稳定。在pH7．1的底物缓冲体系中，37℃，游离酶Km=0.026mol／L；在间歇振摇下固定化酶的表现Km=0．040mol／L。游离酶最适pH为、8.0固定化酶最适pH为9．0。结论 用甲壳糖固定ACE方法简便可行，且固定后的甲壳糖性能稳定，有广阔的应用前景。     

Inhibition of bovine trypsin by the phosphorylated N-terminal part β(1–105) of β-casein

The sensitivities of the R₂₅-I₂₆ bond on bovine β-casein and on its N-terminal fragment β(1–105) to trypsin digestion were compared by monitoring the liberation of the β(1–25) product. It was shown that this peptide bond was poorly and slowly hydrolysed on β(1–105), while it is highly susceptible to trypsin attack when whole protein is used as substrate. The marked resistance of β(1–105) is linked to its inhibitory effect on trypsin activity (apparent K′_i= 1.2 × 10^?6, M), as demonstrated by using a related chromogenic substrate. Indeed, a preincubation step of trypsin with β(1–105) leads to a more pronounced inhibitory effect. The progress curves obtained with and without preincubation show that β(1–105) acts as a slow binding inhibitor on trypsin activity. These findings promise further insight into the action and the regulation of proteolytic enzymes. © Munksgaard 1997.     

Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid

20 chronic periodontitis patients were given a full periodontal examination, including measurements of probing depth, clinical attachment loss, gingival index, bleeding index and plaque index. At a second visit, gingival crevicular fluid (GCF) was collected from the deepest accessible probing site of each tooth. The patients then received scaling, root planing and other appropriate nonsurgical treatment. GCF was collected from the same sites as sampled pretreatment and clinical parameters were measured again. Cathepsin B/L-, elastase-, tryptase-, trypsin-, and dipeptidyl peptidase IV-like activities in GCF samples were determined by fluorimetric assay with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin. Following treatment, there were reductions in all clinical parameters and all protease activities. Most were statistically significant both on a patient level using average patient values and on a site level using either individual patient or pooled patient data. As in previous pre-treatment comparisons, post-treatment protease levels correlated positively and significantly with the corresponding clinical parameters at patient and site levels. The reductions and correlations were more marked for total enzyme activities than concentrations. GCF protease levels appear to reflect the clinical status of periodontal lesions and may thus be of value in monitoring disease activity.     

Isolation of a 33-kDa protein antigen from delipidified Mycobacterium tuberculosis H37Rv

A 33-kDa protein (TB33) was isolated from a delipidated cell sonicate (CS) of Mycobacterium tuberculosis H₃₇Rv (grown in Middlebrook 7H9 broth supplemented with glucose) using immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (Ni-NTA) column. TB33 could not be isolated from the culture filtrate (CF) of M. tuberculosis H₃₇Rv using Ni-NTA. TB33 was recognized by monoclonal antibodies (mAb) known to react with proteins of M. tuberculosis with a molecular mass of 33/34 kDa; namely, mAb F126-5, F67-1 and F126-2. The N-terminal amino acid sequence of TB33 was found to be Xaa-Xaa-Thr-Pro-Ala-Asp-Val-Ser/Cys-Asn-Val-Ala-Ile and thus, shows identity with the N-terminal of antigen 84 of M. tuberculosis except for two mismatches. Antibodies to TB33 could be raised in mice by administering four injections of TB33 (40 μg total protein). Sera from tuberculosis patients reacted with TB33, while those from normal healthy individuals did not. Received: 17 April 1996