PURINE NUCLEOSIDE PHOSPHORYLASE [PNPLⅡ] (T-69)

T-69 PDF download
(Diagnostic Reagent Grade) Nagase Diagnostics ENZYMES T-69REACH適合品

PURINE NUCLEOSIDE PHOSPHORYLASE [PNPLⅡ]

from Bacillus sp.
(Purine–nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1)

Inosine + Pi → Hypoxanthine + D–Ribose 1–phosphate

Preparation and Specification

Appearance
: Colorless to light brownish solution
Specific activity
: More than 500 U/ml

Properties

Molecular weight
: 280±5 kDa (gel filtration)
36±5 kDa (SDS–PAGE)
Isoelectric point
: pH 5.3±0.2
Optimum pH
: 8.0Figure 1
pH stability
: 6.0–10.0 (37℃, 60 min) Figure 2
Optimum temperature
: 65℃ (Tris–HCl buffer) Figure 3
Thermal stability
: Stable at 65℃ and below (pH 8.5, 10 min) Figure 4
Effect of metal ions
: See Table 1

Applications for Diagnostic Test

This enzyme is useful for enzymatic determination of inorganic phosphate when coupled with xanthine dehydrogenase (T–134) .

 PNPL Ⅱ
Pi + Inosine→Hypoxanthine + Ribose 1-phosphate
 XDH Ⅱ
Hypoxanthine + 2 NAD＋+ H2O→Urate + 2 NADH + 2 H＋

 

Table 1. Effect of metal ions on PNPL Ⅱ activity

Metal ionRelative activity
(%)
None100
KCl (10mM)107
NaCl (10mM)107
CsCl (10mM)105
LiCl (10mM)107
NH4Cl (10mM)107
MgCl2 (1mM)107
CaCl2 (1mM)101
BaCl2 (1mM)105
MnCl2 (1mM)36.0
ZnCl2 (1mM)102
CoCl2 (1mM)24.0
CuCl2 (1mM)1.0
NiCl2 (1mM)86.0
EDTA (1mM)112

Fig.1 pH Optimum


〇: Acetate buffer
●: PIPES buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer

Fig.2 pH Stability


37℃, 60min
〇: Acetate buffer
●: Phosphate buffer
□: Tris-HCI buffer
■: Glycine-NaOH buffer

Fig.3 Optimum Temperature


pH 8.0
100mM Tris-HCI buffer

Fig.4 Thermal Stability


pH 8.5, 10min.
100mM Tris-HCI buffer

Assay

Principle

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

 PNPL Ⅱ
Inorganic phosphate+Inosine→Hypoxanthine+D–Ribose 1–phosphate
 XDH Ⅱ
Hypoxanthine+NAD+→Xanthine+NADH+H+
 XDH Ⅱ
Xanthine+NAD++H2O→Urate+NADH+H+

XDH Ⅱ: Xanthine dehydrogenase
NAD : Nicotineamido adenine dinucleotide
Unit definition

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

Reagents
  1. Reaction mixture
    0.2 M Tris–HCl buffer　pH 8.2
    		1.50 ml
  1. 10 mM NAD solution0.45 ml
    30 mM KH2PO4solution0.30 ml
    20 mM Inosine solution0.60 ml
    80 U/ml XDH Ⅱ solution 1) 0.03 ml
    Distilled water0.12 ml
    1) :80 U/ml XDH Ⅱ solution
    Dissolve 800 U of XDH Ⅱ with 10 ml of 20 mM Tris–HCl buffer pH 8.0 containing 5 mM EDTA.
  2. Enzyme dilution buffer
    20 mM Tris–HCl buffer pH 7.5
  3. Reagents
    NAD: NACALAI TESQUE, INC. #24334–84
    Inosine: FUJIFILM Wako Pure Chemical Corporation
    #099–00231
    EDTA: ( 2 Na・2 H2O) KISHIDA CHEMICAL Co., Ltd.
    #060–29133
    XDH Ⅱ : Nagase Diagnostics Co., Ltd.　#T–134
    EDTA: Ethylenediamine tetraacetic acid
Enzyme solution

  1. Dilute accurately 0.5 ml of the sample with enzyme dilution buffer to make a 50–fold solution. Dilute with enzyme dilution buffer to adjust the concentration as required.

Procedure
  1. Pipette accurately 3.0 ml of reaction mixture into a small test tube and preincubate at 37℃.
  2. After 5 min, add 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.
  3. After starting the reaction, measure the rate of increase per minute in absorbance at 340 nm. The rate must be measured within the linear portion of the absorbance curve.
    Absorbance sample :As/min
    blank :Ab/min
    △A/min = (As/min－Ab/min) ≦ 0.045 Abs/min
Calculation
Activity (U/ml) = {(△A/min) /(6.22×2)}× 3.05/0.05 × D
  1. 6.22 :millimolar extinction coefficient of NADH at 340 nm
    ( cm2 /μmole)
    2 :a multiplier derived from the fact that 1 mole of inorganic phosphate produces 2 mole of NADH.
    3.05 :final volume (ml)
    0.05 :volume of enzyme solution (ml)
    D :times of dilution in enzyme solution
Storage

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

PNPL Ⅱ活性測定法 (Japanese)

試薬液
  1. 反応試薬混合液
    0.2M トリス–HCl 緩衝液 pH8.21.50 ml
    10mM NAD 溶液0.45 ml
    30mM リン酸一カリ溶液0.30 ml
    20mM イノシン溶液0.60 ml
    80U/ml XDH Ⅱ溶液1) 0.03 ml
    精製水0.12 ml
    1) :80U/ml XDH Ⅱ溶液
    XDH Ⅱ 800 単位 (U) を5mM EDTA を含む20mM トリス–HCl 緩衝液pH8.0 10ml で溶解する。
  2. 酵素溶解希釈用液
    20mM トリス–HCl 緩衝液 pH7.5
  3. 試薬
    NAD (ニコチンアミドアデニンジヌクレオチド)
    ：ナカライテスク製　#24334–84
    イノシン (Inosine) ：
    富士フイルム和光純薬製　#099–00231
    EDTA (エチレンジアミン四酢酸・2Na・2H2O) ：
    キシダ化学製 #060–29133
    XDH Ⅱ (キサンチン脱水素酵素) ：
    ナガセダイアグノスティックス製　#T–134
酵素試料液
  1. 検品0.5ml を酵素溶解希釈用液で50 倍に希釈する。
  1. その液を酵素溶解希釈用液で適宜希釈する。
測定操作法
  1. 小試験管に反応試薬混合液3.0ml を正確に分注して37℃で予備加温する。
  2. 5 分経過後、酵素試料液50 μl を加えて混和し、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50 μl を加える。
  3. 反応開始後、340nm における吸光度を測定して直線的に反応している1 分間当たりの吸光度変化を求める。
    求められた吸光度変化を試料液はAs/min、盲検液はAb/min とする。
    ΔA/min = (As/min－Ab/min) ≦ 0.045 Abs/min
計算
活性 (U/mg) = {(ΔA/min)/ (6.22 × 2)}×3.05/0.05×D
6.22 :NADH の340nm におけるミリモル分子吸光係数
(cm2 / μmole)
2 :無機リン1 モルからNADH 2モルが生成することによる係数
3.05 :反応総液量 (ml)
0.05 :反応に供した酵素試料液量 (ml)
D :酵素試料液の希釈倍率
T-69 PDF download