Abstract
재료 1.
약재 1)
시약 2)
실험방법 2.
검액 조제 1)
효소 활성도의 측정 2)
단백질 정량 3)
전기영동 분석과
4) Fibrin zymography
효소의 특성 5)
효소 활성도의 측정 1.
Fig. 1. SDS-polyacrylamide gel electrophoresis of total soluble proteins concentrated with 50-75% ethanol from Holotrichia. M lane, protein molecular weight marker; S, sample of Holotrichia.
5 10 15 25
35 75 150
50 100 225 kDa
M
S
Fig. 2. Protease activities of total soluble proteins concentrated with 50-75% ethanol from Holotrichia. Sample 1, freezedry sample of live Holotrichia; sample 2, ustulation samples. One unit of azocaseinolytic activity is defined as the amount of enzyme which causes a net increase of 0.1 in absorbance at 366 nm in 1 hour.
0 1 0 2 0 3 0
4 0 S a m p l e 1
S a m p l e 2
To tal p
rote in (mg)
Azo casein
oly tic activ
ity (U/m
g pr ote in)
Fib rinoly
tic activ ( Ø ity
cm)
혈전분해 효소활성 2.
및
3. SDS-PAGE fibrin zymography
각종 저해제에 의한 영향
4. protease
Fig. 3. Fibrinolytic activities on the fibrin agarose plate of Holotrichia. Upper figure presents fibrin agarose plate and lower figure presents fibrinolytic activities. a, 25 mM sodium phosphate buffer(pH 7.4) as negative control; b, plasmin as positive control;
c, Holotrichia sample.
0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5
FibrinolyticActivity (Øcm)
C o n t r o l P l a s m i n S a m p l e
a b c
Fig. 4. SDS-polyacrylamide gel electrophoresis and fibrin zymography of Holotrichia. M, lane of protein molecular weight markers; 1, 5 ; 2, 10 ; 3, 20 . Zymogen analysis of purified protease for activity staining. Electrophoresis was performed with 8%
native gel containing 0.8% fibrinogen and 100 NIH U/ thrombin. Activities were detected by incubation at 37 for 12hours.
B
M 1 2 3 M 1 2 3
5 15 25 50 100 225
A
Table 1. Effect of Various Inhibitors on the Azocaseinolytic Activities of the Protease of Holotrichia
Fig. 5. Effect of various inhibitors on the fibrinolytic activities of the protease of Holotrichia. The protease was incubated in 25 mM sodium phosphate buffer(pH 7.4) at 37 for 1 hour with various protease inhibitors.
Azocaseinolytic activities were determined as described in the materials and methods section. One unit of azocaseinolytic activity is defined as the amount of enzyme which causes a net increase of 0.1 in absorbance at 366 nm in 1 hour.
None APM
SF(1m M) PM
SF(1m M) TLC
K(1mM ) TPCK
(1mM ) EDTA
(5m M) EGTA
(5mM ) Apr
otinin(1m M) Pep
statinA(1mM ) 0
20 40 60 80 100 120
Relative Activity (%)
