Heat-induced structural changes and aggregation of walleye pollack myosin in the light meromyosin region

Heat-induced structural changes and aggregation properties of walleye pollack myosin, light meromyosin (LMM) and heavy meromyosin (HMM) were investigated. According to the circular dichroism (CD) measurement, the α-helix content of the pollack myosin and LMM were estimated to be 72% and 90% at 5°C but decreased to 22% and 21% by increasing the temperature to 60°C with two transitions at 35°C and 50°C, respectively. In contrast, that of HMM decreased gradually from 37% to 33% by increasing the temperature from 5°C to 40°C, and decreased steeply to 20% above 50°C. These results indicate that the decrease in the α-helix content in the myosin molecule upon heating was attributable mainly to the decrease in the α-helix content in the LMM region. In contrast, 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence and light scattering intensity of both myosin and HMM were remarkably increased above 25°C and 35°C, respectively, while those of LMM showed only a slight change even above 60°C. Although LMM alone formed no aggregates detectable by the light scattering measurement, it formed coprecipitates with myosin but not with HMM upon heating at 40°C for 10 min. These facts suggest that LMM bind to the LMM region of the myosin. Further, it was found that myosin gel formed in a test tube by the same heating conditions was significantly weakened by coexistence of LMM. These results suggest that the association of the LMM region of myosin molecules is essential for the heat-induced gelation of myosin.     

Measurement of Carbonic Anhydrase I and II Isozymes in Feces as a Marker of Occult Blood in Horses with Intestinal Tract Bleeding

Although endoscopy is the definitive diagnostic method for the detection of colonic ulcers, the equipment required for performing the test is costly and difficult to use. Therefore, a simple cost-effective and reliable screening test for intestinal tract bleeding is needed. To this end, we measured carbonic anhydrase isozymes (CA-I and CA-II) originating from erythrocytes by ELISA in order to determine if they could be used as markers of occult blood in feces. For fecal extract preparation, 2 g of feces were mixed with 4 ml of 0.01 M Tris-HCl (pH 8.0) containing 0.01% thimerosal. The concentrations of CA-I and CA-II in the fecal samples of 13 clinically normal racehorses were found to be 30.0 ± 10.0 and 34.0 ± 13.0 ng/ml, respectively. Increased concentrations of CA-I were detected in the fecal samples of 5 horses after blood administration; however, no increase was observed in CA-II. The concentrations of CA-I and CA-II in the fecal samples of 88 racehorses with clinical signs of equine gastric ulcer syndrome (EGUS) were 115.3 ± 79.0 and 41.0 ± 42.0 ng/ml, respectively. Thus, our results indicate that CA isozymes can be useful as markers of occult blood in the fecal samples of horses with intestinal tract bleeding.     

Measurement of carbonic anhydrase isozyme VI (CA‐VI) in swine sera,colostrums, saliva,bile, seminal plasma and tissues

Swine secretory carbonic anhydrase VI (CA‐VI) was purified from swine saliva and an antibody to CA‐VI was generated. A specific and sensitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the measurement of swine CA‐VI. The assay can detect as little as 5 ng/mL of swine CA‐VI. Typical standard curves were determined for a range of CA‐VI solutions (7.8 to 500 ng/mL). The coefficients of variation for these solutions were less than 5%. When 500, 250 or 100 ng/mL of swine CA‐VI was added to swine sera, the recoveries were 102.0%, 109.7% and 100.2%, respectively. The concentrations of CA‐VI in the saliva (26.2 ± 30.4 µg/mL), sera (3.3 ± 4.9 ng/mL), bile (153.0 ± 114.0 ng/mL), seminal plasma (124.0 ± 39.0 ng/mL) and parotid gland (441.3 ± 90.0 µg/g wet tissue), submaxillary gland (88.1 ± 124.4 µg/g wet tissue), sublingual gland (58.6 ± 24.6 µg/g wet tissue) and gallbladder (2.4 ± 1.3 µg/1g wet tissue) were determined by ELISA. The concentration of CA‐VI in colostrum was 163.3 ± 101.4 ng/mL and did not decrease within 10 days following parturition. An immunohistochemical reaction to anti‐CA‐VI antiserum was observed in the columnar epithelial cells lining the gallbladder. These data suggest that secretory CA‐VI plays various roles in pH regulation and the maintenance of ion and fluid balance.