TABLE 2.
Effect of enzymes on ICAM-1-inducing activity of LPsal and LPfer
| Treatment | Mean ± SD of ICAM-1-inducing activitya(A405)
|
|
|---|---|---|
| LPsal | LPfer | |
| None | 0.99 ± 0.07 (100) | 1.08 ± 0.04 (100) |
| Proteinase K | 1.17 ± 0.09 (118) | 1.16 ± 0.04 (107) |
| Endoglucosidase H | 1.07 ± 0.06 (108) | 1.11 ± 0.03 (103) |
| Endoglucosidase D | 1.05 ± 0.06 (106) | 1.14 ± 0.03 (115) |
| Lipoprotein lipase | 0.08 ± 0.01 (8) | 0 |
Two hundred microliters of Lpsal (173 μg of protein/ml of PBS) or Lpfer (113 μg of protein/ml of PBS) was treated at 37°C for 2 h with 0.2 μg of each enzyme, and then 20 μl of the reaction mixture was added to the monolayer of Gin-1 cells grown in 200 μl of DME medium in each well of a 96-well flat-bottomed microplate. After a 6-h incubation, ICAM-1 expression was measured by Cell-ELISA. Enzyme and buffer controls were run at the same time. Absorbance at 405 nm of enzyme or buffer control was the same as that for the control medium. Therefore, A405 was expressed as the absorbance at 405 nm of each well − the mean absorbance at 405 nm of three medium control wells). Data, expressed as means ± standard deviations from triplicate wells, are representative of three separate experiments. Values in the parentheses are 100 × (mean A405 of a specimen treated with enzyme/mean A405 of a specimen left untreated).