Establishment Of β-D-Galactosidase Activity Assay Method And Change Of Its Activity In Digestive Tract Tumor

Objective:To investigate the differential profile of P-D-galactosidase (GAL) activity in digestive tract mucosa tissues and the change of GAL levels in tumor tissues,we established a simple method to measure GAL activity and detected GAL activity in various digestive tract mucosa tissues.Methods: 1. Establishment of method for GAL activity assay: (l)Preparation for enzyme extract: The digestive tract mucosa tissues were washed with phosphate buffer (lOmmol/L, pH6.5), minced and homogenized. The enzyme extracts were collected by centrifugation . The protein concentration was determined by modified Lowry assay using bovine serum albumin as standard. (2) GAL activity assay: The final concentrations of components in 1ml of reaction mixtures were as follows: 0.8 u mol o-nitrophenyl 0 -D-galactopyranoside (ONPG) in O.lmol/L citrate buffer (pH4.5) and 2mg protein. of enzyme extract. After incubation for 30 min. at 37C,the reactions were stopped by addition of 1ml 0.5mol/L sodium carbonate. The absorbances at 405nm were read. Enzyme activity was expressed in U/mg protein.2. Changes of GAL activity in digestive tract carcinoma tissues: 31 cases with digestive tract tumors operated were selected, in which 8 cases were rectal adenocarcinomas, 6 cases were colonic adenocarcinomas, 6 cases were cardiac adenocarcinoma, 5 cases were gastric adenocarcinoma, 6 cases were esophageal squamous carcinoma. Assays of GAL activity in mucosa enzyme extract were performed using the method.3. MMP-9 relative activity assay in digestive tract mucosa tissues: MMP-9 relative activity was detected in rectum(6cases), colon(5 cases), cardiac(6 cases),gastric(5 cases), esophagus (6 cases) of the normal and the tumor tissues using gelatin-zymography assay according to previously described.Results: 1. Establishment of method for GAL activity assay: The GAL activity was measured using this method. The results showed that the variability within batchs was 3.92%~4.89% and the variability between batchs was 3.8%. These suggested that the method was so reliable that it could be used in GAL assay. The method had the merits of high precision, good reproducibility and was easy to operate, so it could detect GAL activity successfully.2. Changes of GAL activity in digestive tract carcinoma mucosa: The results showed that GAL activity could be detected in digestive tract normal mucosa tissues and it was higher in colon(0.47?.05)-rectal(0.43?.03) mucosa thanthat in other mucosa tissues. In the cases of colon(0.60?.06)-rectal (0.77?.07) carcinoma group the GAL activity was significantly higher than that of the normal(P<0.05).3. Changes of MMP-9 relative activity in digestive tract tumor tissues: The results suggested that extract of digestive tract carcinoma tissues mainly contained 92kD gelatinase B(MMP-9). MMP-9 relative activity in any digestive tract tissue of the carcinima group (colon 88.00?.72> esophagus 64.17?.90> cardia 64.00?.73 >gastric 50.80?.20 >rectum 46.00?.18)was much higher than that of the normal mucosa (cardia 52.00?.49> colon 50.80?.98> rectum 38.33?.04> gastric 31.00?.65> esophagus 26.67?.19)(P<0.05).Conclusion: The results suggested that GAL and MMP-9 activities could be detected in digestive tract normal mucosa. It might be correlated with the digestion and absorption of carbohydrate and the renew activation of glandular cell. Their activities in tumor tissues were significantly higher than that of the normal. According to the fact that GAL and MMP-9 play key roles in the modification of cell surface carbohydrate composition and the remodelling of extracellular matrix(ECM), we concluded that they might be involved in the invasion and metastasis of digestive tract carcinoma.