(Diagnostic Reagent Grade) ASAHI KASEI ENZYMES T-67REACH適合品

NAD SYNTHETASE [NADSⅡ]

from Geobacillus stearothermophilus
(Deamido–NAD+ : ammonia ligase (AMP–forming) , EC 6.3.1.5)
(NAD synthase)

Deamido–NAD⁺ + ATP + NH₃ → NAD⁺ + AMP + PPi

Preparation and Specification

Appearance: : White amorphous powder, lyophilized

Specific activity: : More than 1.00 U/mg solid

Properties

Substrate specificity: : See Table 1

Molecular weight: : 50 kDa (gel filtration)
25 kDa (SDS–PAGE)

Michaelis constants: : Deamido–NAD 2.4 × 10^-5M
ATP 4.3 × 10^-5M
NH₃ 2.16 × 10^-3M

Isoelectric point: : pH 5.2 ± 0.2

Optimum pH: : 9.0–10.5Figure 1

pH stability: : 6.0–9.0 (37℃, 15 min) Figure 2

Optimum temperature: : 70℃ (Tris–HCl buffer) Figure3

Thermal stability: : Stable at 60℃ and below (pH 7.5, 10 min) Figure4

Effect of metal ions: : See Table 2

Effect of detergents: : See Table 3

Applications for diagnostic Test

This enzyme is useful for enzymatic determination of ATP, ammonia, urea or creatinine when coupled with creatinine deiminase.
This enzyme is suitable for enzymatic cycling method when coupled with dehydrogenase and diaphorase (T–06) .
Please refer to an information 3α–hydroxysteroid dehydrogenase (T–58)

	UR
Urea + H₂O	→	2 NH₃ + CO₂
	NADS Ⅱ
Deamido-NAD^＋ + AＴＰ + NH₃	→	NAD^＋ + AMP + PPi

UR: Urease

Table 1. Substrate specificity

Substrate (1mM)	Relative activity (%)
NH₄CI	100
L–aminoacids (Leu, Arg, Lys, Ileu, Tyr, Ala, Glu, Gln, Asn, Gly, Ser)	0
Azaserine	0
Urea	0
Uric acid	0
Creatinine	0
Creatine	0
Tris	0
Good's buffers	0

Table 2. Effect of metal ions on NADS Ⅱ activity

Metal ion (1mM)	Relative activity (%)
None	100
NiCl ₂	1.1
BaCl ₂	106.0
SnCl ₂	99.8
AlCl ₃	95.6
CdSO ₄	2.4
MnCl ₂	18.2
CuCl ₂	3.6
ZnCl ₂	5.8
CoCl ₂	94.2
MgCl ₂	102.0
CaCl ₂	103.0
HgCl ₂	0.3

Table 3. Effect of detergents on NADS Ⅱ activity

Detergent (0.1%)	Relative activity (%)
None	100
Adekatol SO–120	108.2
SO–145	106.9
Brig 35	107.5
Cation DT–205	10.5
FB	11.8
Cetylpyridinium chloride	1.6
Sodium dodecyl sulfate	2.3
Tween 80	102.3
Cholic acid	105.6
Cetyltrimethyl ammonium chloride	4.2
Span 85	104.6
Sodium laurylbenzene sulfonate	6.5

Fig.1 pH Optimum

〇: 3,3-Dimethylglutarate-NaOH buffer
●: Tris-HCI buffer
□: Glycine-NaOH buffer

Fig.2 pH Stability

37℃, 15min
〇: 3,3-Dimethylglutarate-NaOH buffer
●: Tris-HCI buffer
□: Glycine-NaOH buffer

Fig.3 Optimum Temperature

pH 8.0
100 mM Tris-HCI buffer

Fig.4 Thermal Stability

pH 7.5, 10min.
100 mM Tris-HCI buffer

Assay

Principle

The assay is based on the increase in absorbance at 340 nm as the formation of NADH proceeds in the following reactions:

	NADS Ⅱ
Deamido–NAD+NH₄⁺+ATP	→	NAD⁺+AMP+PPi
	Mg²⁺
	3α–HSD Ⅱ
NAD⁺+Cholic acid	→	3 –Oxo–cholic acid+NADH

NAD:Nicotineamido adenine dinucleotide
ATP:Adenosine triphosphate

Unit definition

One unit is defined as the amount of enzyme which converts 1 μmole of deamido–NAD to NAD⁺ per minute at 37℃ under the conditions specified in the assay procedure.

Reagents

Reaction mixture

1 M Diethanolamine–HCl buffer pH 9.5	0.20 ml
20 mM Deamido–NAD solution	0.20 ml
0.2 M ATP solution pH 7.0	0.15 ml
0.1 M MgCl₂ solution	0.30 ml
0.1 M NH₄Cl solution	0.30 ml
200 U/ml 3α–HSD Ⅱ solution	0.20 ml
50 mM Sodium cholate solution	0.40 ml
Distilled water	0.25 ml

Reaction stopper
0.3 M EDTA solution pH 9.5
EDTA: Ethylenediamine tetraacetic acid
Enzyme dilution buffer
20 mM Bicine–NaOH buffer pH 8.5
Reagents
Diethanolamine:
FUJIFILM Wako Pure Chemical Corporation
Special grade #093–03115
Deamido–NAD: Oriental Yeast Co., Ltd.
ATP: Kyowa Hakko Co., Ltd.
3α–HSD Ⅱ: Asahi Kasei Pharma Corporation　#T–58

Sodium cholate: Tokyo Kasei Kogyo Co., Ltd.
Special grade　#C 0325
EDTA (2Na・2H₂O) : KISHIDA CHEMICAL Co., Ltd.
#060–29133
Bicine: Dojindo Laboratories　#347–03282

Enzyme solution

Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration to within 0.3–0.7 U/ml.

Procedure

Pipette accurately 2.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
After 5 min, add exactly 50 μl of enzyme solution and mix to start the reaction at 37℃.

※ In the case of a test blank, add 50 μl of enzyme dilution buffer in place of enzyme solution.
At 5 min after starting the reaction, add 1.0 ml of the reaction stopper to stop the reaction.
Measure the absorbance at 340 nm.

Absorbance sample : As

blank : Ab

0.130Abs ≦ △A = (As−Ab) ≦ 0.390 Abs

Calculation

Activity (U/mg of powder) = {(△A/5)/6.3} × 3.05/0.05 × 1/x

6.3:	millimolar extinction coefficient of NADH at 340 nm ( cm² /μmole)
5 :	reaction time (min)
3.05 :	final volume (ml)
0.05 :	volume of enzyme solution (ml)
X :	concentration of the sample in enzyme solution ( mg/ml)

Storage

Storage at−20℃ in the presence of a desiccant is recommended.

NADS Ⅱ　活性測定法 (Japanese)

試薬液

反応試薬混合液

1.0 M DEA (Diethanolamine) –HCl 緩衝液 pH9.5	0.20 ml
20 mM デアミド–NAD (NaNAD) 溶液 ¹⁾	0.20 ml
0.2 M ATP 溶液 pH7.0 ²⁾	0.15 ml
0.1 M MgCl₂ 溶液	0.30 ml
0.1 M NH₄Cl 溶液	0.30 ml
200 U/ml 3α–HSD Ⅱ溶液 ³⁾	0.20 ml
50 mM コール酸ナトリウム溶液	0.40 ml

精製水

0.25 ml

1) :	20mM デアミドNAD 溶液デアミドNAD 133 mg (純度換算) を精製水10 ml で溶解する。
2) :	0.2M ATP 溶液 pH7.0 ATP1.21g を精製水8ml に溶解した後、4N NaOH でpH7.0 に調整し、精製水で全容を10ml とする。
3) :	200U/ml 3α–HSD Ⅱ溶液 3α–HSD Ⅱ 2,000U を精製水10ml で溶解する。

反応停止液
0.3 M EDTA 溶液 pH9.5
酵素溶解希釈用液
20 mM Bicine–NaOH 緩衝液 pH8.5
試薬
DEA (ジエタノールアミン) ：
富士フイルム和光純薬製　特級　#093–03115
デアミドNAD：オリエンタル酵母製
ATP (アデノシン・三リン酸・2Na・3H₂O ) ：
協和発酵製
3α–HSD Ⅱ：旭化成ファーマ製 #T–58
コール酸ナトリウム：
東京化成工業製　特級　#C 0325
EDTA (エチレンジアミン四酢酸・2Na・2H₂O ) ：
キシダ化学製 #060–29133
Bicine (ビシン) ：同仁化学製　#347–03282

酵素試料液

検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml とする。
その液を酵素溶解希釈用液で0.3–0.7U/ml 濃度となるように適宜希釈する。

測定操作法

小試験管に反応試薬混合液2.0ml を正確に分注し、37℃で予備加温する。

5 分経過後、酵素試料液50 μ1 を正確に加えて混和し、37℃で反応を開始する。

※ 盲検は酵素試料液の代わりに酵素溶解希釈用液50μ1 を加える。
5 分経過後、反応停止液1.0ml を加えて混和し、反応を停止する。
340nm における吸光度を測定する。
求められた吸光度変化の試料液はAs、盲検液はAbとする。
0.130Abs ≦ ΔA = (As−Ab) ≦ 0.390 Abs

計算

活性 (U/mg) = {(ΔA/5)/6.3} × 3.05/0.05 × 1/x

6.3 :	NADH の340nm におけるミリモル分子吸光係数 (cm² /μmole)
5 :	反応時間 (min)
3.05 :	反応総液量 (ml)
0.05 :	反応に供した酵素試料液量 (ml)
X :	酵素試料液中の検品濃度 (mg/ml)

Absorbance sample :	As
blank :	Ab

NAD SYNTHETASE [NADSⅡ] (T-67)