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Hydrolysis of ceramide by ceramidase (CDase) and measurement of CDase activity

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Introduction Protocol References Credit lines
Category
Glycosidases & related proteins
Protocol Name

Hydrolysis of ceramide by ceramidase (CDase) and measurement of CDase activity

Authors
 Nozomu Okino *
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
Keywords

ceramidase

ceramide

sphingosine

Reagents
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Ceramidase (CDase)

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C12-NBD-Cer (Avanti)

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TLC plate (Silicagel 60, 20 x 20 cm, Merck)

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chloroform (CHCl3)

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methanol (MeOH)

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CHCl3-MeOH mixture (C/M, volume/volume)

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Ninhydrin reagent:

Dissolve 250 mg of ninhydrin in 100 ml of water-saturated n-butanol. Store the reagent in a refrigerator.

Instruments
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TLC developing chamber (inside, ~24 cm x ~11 cm x ~21 cm)
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Hot plate
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TLC Chromatoscanner (CS-9300, Shimadzu)
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UV transiluminator or AE-6935B Visirays (ATTO)
Methods
1.

Hydrolysis of ceramide by CDase from Pseudomonas aeruginosa

1) 

 Dissolve the ceramide (~10 nmol) in 10 µl of 50 mM Tris-HCl buffer, pH 8.5, containing 1.0%(w/v) Triton X-100 and 5 mM CaCl2.

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2) 

 Add 10 µl of Pseudomonas CDase (~1 mU) and incubate at 37oC for an appropriate period.

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3) 

 Stop the reaction by adding 50 μl of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge at 16,000 x g for 1min.

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4) 

 Remove the upper phase and dry the lower phase.

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5) 

 Dissolve the sample in 20 μl of chloroform/methanol (2:1, v/v) and apply to a TLC plate.

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6) 

 Develop the TLC plate with chloroform, methanol, and 25% ammonia (14:6:1, v/v/v) in a TLC chamber.

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7) 

 Dry the plate, heat on a hot plate at 110°C for 5 min, and spray on the ninhydrin reagent. Ninhydrin reagent can detect free amino groups of sphingosine derived from the enzymatic reaction.

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2.

Detection of CDase activity from Pseudomonas aeruginosa

1) 

 Dissolve 1 nmol of C12-NBD-Cer in 10 μl of 2x reaction buffer (50 mM Tris-HCl buffer, pH 8.5 containing 0.5%(w/v) Triton X-100 and 5 mM CaCl2).

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2) 

 Add 10 μl of CDase and incubate at 37oC for an appropriate period (~1 h).

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3) 

 Stop the reaction by adding 50 μl of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge at 16,000 x g for 1min.

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4) 

 Apply 5 μl of the lower phase to a TLC plate.

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5) 

 Develop the TLC plate with chloroform, methanol, and 25% ammonia (14:6:1, v/v/v) in a TLC chamber.

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6) 

 Dry the plate and visualize C12-NBD-Cer and C12-NBD-fatty acid with an AE-6935B Visirays or UV transilluminator. Quantifying the C12-NBD-fatty acid released by the actions of the enzyme and the remaining C12-NBD-Cer with a Shimadzu CS-9300 chromatoscanner (excitation, 475 nm; emission, 525 nm). One enzyme unit of CDase is the amount capable of catalyzing the release of 1 μmol of C12-NBD-fatty acid/min from C12-NBD-Cer.

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3.

Detection of CDase activity from mammalian cells or tissues

1) 

 When mammalian cells or tissues are used for the enzyme source3-5), 50 mM Tris-HCl buffer, pH 7.5 containing 2%(w/v) sodium cholate should be used for the 2x reaction buffer.

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Figure & Legends

Figure & Legends

 

Fig. 1. Action mode of CDase.

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Date of registration：2012-11-13 11:41:45
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